Control marker for implementing analysis methods on spots

ABSTRACT

The present invention relates to the use of a control marker for implementing analysis methods on spots, in particular in the context of multiplex analyses. The present invention thus relates to solid supports containing said control marker, their preparation method and their use in analysis methods. The present invention makes it possible to verify the presence, location and/or integrity of the spots at the end of the analysis method, and thus to secure the obtained results while guaranteeing that the yielded result indeed results from a present, intact and localized spot.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 16/199,509,filed Nov. 26, 2018, now allowed, which is a division of U.S.application Ser. No. 15/302,512, filed Oct. 7, 2016, now U.S. Pat. No.10,139,404, which is the U.S. national stage application ofInternational Patent Application No. PCT/EP2015/057639, filed Apr. 8,2015.

FIELD OF THE INVENTION

The present invention relates to the use of a control marker forimplementing analysis methods on spots, in particular in the context ofmultiplex analyses. The present invention thus relates to solid supportscontaining said control marker, their preparation method and their usein analysis methods. The present invention makes it possible to verifythe presence, location and/or integrity of the spots at the end of theanalysis method, and thus to secure the obtained results whileguaranteeing that the yielded result indeed results from a present,intact and localized spot.

BACKGROUND OF THE INVENTION

A multiplex analysis method allows the simultaneous detection of thepotential presence of several analytes within a same sample. A multiplexanalysis method is typically implemented using a solid supportcomprising spots, for example a microplate comprising spots in eachwell, the spots each being intended to detect an analyte or to serve asa control.

It is clear for one skilled in the art that one risk related to spottechnology is the absence of deposit, the elimination or deteriorationof several spots during the preparation method for the solid support, inparticular a microplate, or during the implementation of the analysismethod using said solid support. The device for depositing samples orreagents may indeed accidentally come into contact with one or severalspots, thereby altering their surface, for example by forming astriation in one or several spots, or by pulling out all or part of oneor more spots.

For example, in the article by Bastarache et al. (Accuracy andReproducibility of a multiplex Immunoassay platform: a validation study,J. Immunol Methods, Mar. 31, 2011, 367 (1-2) 33-39), flaws are presentedthat were observed at the end of testing on the light signal, such asspot irregularities, the presence of comas that cause the contaminationof one spot by a neighboring spot and the absence of expected signal atthe theoretical location of the spot.

Yet when a negative result is rendered at the end of the analysismethod, this result must result from the absence of the analyte to bedetected in the sample, and not from an absence or a deterioration ofthe detection spot of the corresponding analyte. Securing analysismethods is crucial, in particular for their use in diagnostics inhumans, for example to verify the absence of viral or bacterialcontamination of a blood sample for transfusion purposes.

The manufacture of a solid support for a spot analysis consists ofdepositing, on the surface of the solid support, solutions comprising acapture ligand of the analyte to be detected, so as to form spots. Thequality of the solid support is next verified at the end of themanufacturing of the solid support, so as to keep only the solidsupports having intact and well-formed spots.

Thus, document US2006/0063197 describes the use of a fluorophore in thedeposition solution intended to form the spots of a DNA microarray. Thefluorophore is used to verify the quality of each spot at the end of thepreparation method for the DNA microarray. Furthermore, only a weakresidual fluorescence signal is detected at the spots before adding thesubstrate, during the implementation of an ELISA test.

Document WO2012/142397 examines flux issues in microfluidic apparatusescontaining DNA microarrays and describes a DNA microarray assemblycomprising an array chamber with an inlet for the sample at a first end,a DNA microarray and an outlet for the sample at a second end connectedto a waste chamber, the area transverse to the first end of the arraychamber being wider than that at the second end. This document alsodescribes a method for verifying the manufacturing quality of a DNAmicroarray, by measuring the fluorescence emitted by an internal qualitycontrol fluorophore at the spots of an array and encoding theinformation relative to each spot of the array in a barcode, a memorydevice or by radiofrequency identification, this information thusencoded being associated with the DNA microarray.

Furthermore, during the implementation of a multiplex analysis method,the signal corresponding to the analyte to be detected can be detectedat a theoretical reading grid. This theoretical reading grid isgenerally defined at the end of the multiplex analysis method, from thesignal detected at a spot serving as a positive control. However, inlight of the reading scales, any shift in the positioning of the spotsduring the manufacture of the solid support and/or in the positioning ofthe solid support in the device for detecting the signal relative to thetheoretical reading grid causes a shift in the actual positioning of thespots relative to the theoretical reading grid and therefore affects thedetection sensitivity of the analytes.

There is therefore still a need for solutions to secure the resultsobtained at the end of an analysis method on spots, in particular bymaking it possible to verify the presence, location and/or integrity ofthe spots at the end of the analysis method and/or by making it possibleto optimize the detection of analytes, for example by improving thedetection sensitivity of the analytes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on revealing control markers that may bedeposited, to form spots on a solid support, at the same time asspecific capture ligands of the analytes to be detected in a sample,these control markers being capable of producing a signal at the end ofan analysis method implemented using said solid support, interferingnegligibly or not at all with the detection of the analytes. The controlmarkers according to the invention thus make it possible to verify thepresence, location and/or integrity of the spots at the end of ananalysis method. These control markers are qualified here as “resistantcontrol markers”.

The expressions “analysis method” and “method for detecting at least oneanalyte” are synonymous here.

The terms “produce”, “product” or “production” apply to any type ofsignal, and in particular any type of electromagnetic radiation, whetherit involves a radioactive signal, a light emission or absorbance.

When the detected signal is a fluorescence luminescence signal, the“produced signal” is in particular an “emitted signal”, and “produce asignal” and “production of a signal” then mean “emit a signal” and“emission of a signal”, respectively.

“Detection of at least one analyte” (or “detect at least one analyte”)means, within the present application, detection of the presence (ordetect the presence) of said analyte(s) and/or quantification of saidanalyte(s) (or quantify said analyte(s)).

“End of an analysis method” means, within the meaning of the invention,after the spots have been placed in the presence of the sample to beanalyzed, a ligand or ligands for detecting an analyte, if applicable atleast one (first) reporter of a detection marker coupled to a detectionligand of an analyte and, if applicable, at least one (second) reporterof a detection marker coupled to said first reporter.

Surprisingly, the inventors have thus shown resistant control markersthat can be fixed on a solid support, that do not interfere with theanalysis method itself and that remained completely detectable (inparticular because they remain at least partially fixed on the solidsupport) after implementing different steps of the analysis method. Whenthe signal produced by the resistant control marker at the end of theanalysis method makes it possible to define a spot meeting the qualitycriteria of a spot, in particular presence, location and/or integrity,the signal corresponding to an analyte can then be detected at thisspot. In particular, the resistant control markers according to theinvention can be used in an analysis method in which the presence of atleast one analyte is detected by a signal emitted by chemiluminescence,preferably via the reaction of a peroxidase enzyme with a luminolsubstrate and/or an analogue of luminol, for example isoluminol, and/orone of their derivatives.

The resistant control markers according to the invention have theadvantage of making it possible to control spots of a solid support atthe end of an analysis method, and not only at the end of thepreparation method of said solid support.

Thus, the present invention makes it possible to guarantee the resultsfor the user. In particular, the present invention makes it possible toguarantee that a negative result indeed results from the absence of theanalyte in a sample and not for example from an absence of spot or ashift in the reading of the spot. In other words, the present inventionmakes it possible to eliminate false negative results (also called“false negatives”) related to a flaw of a spot (i.e., a spot notcompliant with the quality criteria) and/or a reading flaw of a spot atthe end of the analysis method and that are not detectable in themultiplex analysis methods traditionally used, in which the reading gridis adjusted theoretically, for example on the signal emitted by apositive control spot. The present invention also makes it possible toguarantee that a positive result indeed results from the presence of theanalyte in a sample. For example, a signal may be detected correspondingto the marker of an analyte in the theoretical location of a spot (asdefined by a theoretical reading grid, for example adjusted on thesignal emitted by a control spot), whereas there is no spot in thatlocation; such a falsely positive result (also called “false positive”)would then occur during the implementation of a traditional analysismethod; by using a resistant control marker according to the inventionin the spots of the solid support, no signal corresponding to saidresistant control marker will be detected at this theoretical locationof a spot and the analysis method will lead to an absence of resultrendered, despite the detection of a signal corresponding to the markerof the analyte in that location.

Another advantage of the present invention lies in the fact that theresistant control markers make it possible to define the reading grid atthe end of the analysis method. Yet there may be differences between atheoretical reading grid defined according to the physical parameters ofthe solid support and that defined at the end of an analysis methodusing said solid support. These differences may for example result froma shift between the expected theoretical spot grid and the grid actuallyobtained at the end of the analysis method (one or several spots); thisshift is thus corrected by the device described in the presentapplication. It is thus more reliable to define the reading grid of asolid support at the end of the analysis method, preferably with thesame detection device as that used to detect the signal produced by thedetection marker of at least one detection ligand of an analyte, andpreferably concomitantly. Thus, the resistant control markers accordingto the invention also make it possible to secure the results of ananalysis method on spots, by improving the sensitivity of the analytedetection owing to the definition of a reading grid to detect analytesfrom the location of the spots detected at the end of the analysismethod.

Lastly, the preparation of a solid support whereof the spots comprise aresistant control marker according to the invention is simple to do anddoes not add any additional steps, the resistant control marker forexample being able to be simply mixed with the compound of interest,such as a capture ligand, in the solution to be deposited to form thespot.

The present invention in particular relates to a solid support for asecure detection of at least one analyte in a sample, whereof at leastone spot intended to detect an analyte comprises at least one resistantcontrol marker and at least one capture ligand of an analyte, a methodfor preparing such a solid support and a secure detection method for atleast one analyte in at least one sample using said solid support.

“At least one”, within the meaning of the present application, refers toone or several, several in particular meaning two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen or more than fifteen. Similarly, in the present application, “atleast” means, x or more than x, and in particular x+1, x+2, x+3, x+4,etc., “x” being an integer greater than or equal to 2, for example 2 or3.

One particularly preferred analysis method according to the invention isa multiplex analysis method.

The present invention also relates to a method for selecting a resistantcontrol marker, as well as the use of at least one resistant controlmarker in at least one spot intended to detect an analyte to secure amethod for detecting at least one analyte in a sample.

The present invention also provides an appropriate device for improveddetection of at least one analyte in a sample.

Sample

The sample to be analyzed is preferably a biological sample.

The biological sample may be a biological fluid, such as a sample ofblood, blood derivatives (such as plasma or serum), urine, cerebrospinalfluid, saliva or combinations thereof, or a tissue sample, such as atissue obtained by biopsy, a cell, a set of cells, a plant extract, orcombinations thereof.

A blood derivative refers to any product, in particular fluid, obtainedfrom a blood sample.

The sample to be analyzed may also be a culture medium and/or a culturesupernatant.

Before being analyzed, the sample may undergo one or several priortreatment steps, such as dilution, centrifugation, heat treatment, celllysis, solubilization, and denaturation (for example by one or severalchaotropic agents, one or several reducing agents and/or by heating),extraction, PCR (Polymerase Chain Reaction), addition of an unmarkeddetection ligand or combinations thereof. The addition of an unmarkeddetection ligand is in particular useful to implement a neutralizationtest.

The sample may also be a mixture of at least two samples that may be ofthe same nature or different natures.

Examples of mixtures of samples of different natures are a mixture ofblood and urine, a mixture of blood and plasma, a mixture of serum andplasma, or a mixture of blood, serum and plasma.

One preferred sample according to the invention is a sample or mixtureof samples of blood and/or blood derivatives (in particular plasmaand/or serum).

Analyte

An analyte to be detected in a sample may be any type of compound,natural or synthetic, that one wishes to detect and/or quantify in asample.

An analyte may for example be a protein, a peptide, a glycoprotein, acarbohydrate, a cell, an organelle, a virus or a nucleic acid.

The cell may be an animal cell, a plant cell, a bacteria cell, aprotozoa, a metazoan cell, a yeast cell or a fungus cell.

A nucleic acid designates a polymer of nucleotides linked byphosphodiester bonds, such as a deoxyribonucleic acid (DNA), aribonucleic acid (RNA) or an analogue thereof, such as phosphorothioatesor thioesters, in single-strand or double-stranded form.

The analyte or at least one of the analytes is thus for example chosenfrom the group consisting of an antigen, an antibody, an antibodyfragment, a hapten, a hormone, a hormone receptor, an enzyme, or anucleic acid.

The analyte(s) are preferably selected from the group consisting of anantigen, an antibody, an antibody fragment, a hapten, a hormone, ahormone receptor and an enzyme.

Within the meaning of the present application, “antigen” refers to anatural, recombinant or synthetic molecule recognized by antibodies orcells of the immune system and capable, when it is presented underappropriate conditions to the immune system of a host, of inducing animmune response.

An antigen may be a molecule, in particular a polypeptide, comprising orconsisting of at least one epitope that may be linear or conformational.The term “linear epitope” refers to a polypeptide, in particular apeptide, comprising or generally consisting of 3 to 15 amino acids, moregenerally 5 to 15 amino acids, preferably at least 6, 8, 10 or 12 aminoacids, capable of binding to an antibody molecule against said antigen.The term “conformational epitope” refers to a three-dimensionalstructure recognized by an antibody and determined by the juxtapositionof several amino acids in space, which may be noncontiguous in thepeptide sequence of the protein (or polypeptide) against which thisantibody is directed, but which, due to the folding of the polypeptidechain, find themselves dose to one another in space, and can thus form apattern that may be recognized by an antibody.

An antigen within the meaning of the present invention is for example aprotein (in particular a native protein or a recombinant protein), apeptide (for example, a synthetic peptide), a glycoprotein, acarbohydrate or a lipid; said peptide may or may not be associated witha carrier molecule.

A “carrier molecule” in particular refers to a protein or carbohydratecarrier molecule, in particular a carrier protein. A carrier moleculemay be a polypeptide (in particular protein or a peptide), which may ormay not be natural (for example, a recombinant protein or a syntheticpeptide), a functionalized polymer (such as dextran, polysaccharide orpolylysine), a mixed copolymer (in particular a copolymer of differentamino acids, for example a lysine-tyrosine copolymer). The carriermolecule may be an antibody (in particular a monoclonal antibody or apolyclonal antibody), for example an immunoglobulin (also called Ig).

One example carrier molecule or protein is BSA (bovine serum albumin).

“Hapten” in the present application refers to a molecule with a lowmolecular weight capable of being recognized by the immune system, butwhich is immunogenic only when it is coupled to a carrier molecule.

An analyte is preferably a compound making it possible to diagnose acondition in a subject, which may or may not be pathological, or todiagnose the risks of developing a condition, which may or may not bepathological. An example of a non-pathological condition is a pregnancy.

The subject may be a human, a non-human animal or a plant. The non-humananimal is preferably a mammal, such as a cat, dog, monkey, rabbit, mouseor rat.

The term “human” is used broadly and in particular designates a man or awoman of any age, such as an infant, a child, an adolescent, an adult oran elderly person.

In one preferred embodiment, at least one analyte is chosen from amongan antigen or an antibody.

When the analyte or one of the analytes is an antigen, it is preferablyan antigen making it possible to diagnose an infection, for example aninfection caused by a virus, a bacteria, a fungus, a protozoa or ametazoan.

When the analyte or one of the analytes is an antibody, it is preferablyan antibody making it possible to diagnose an infection, for example aninfection caused by a virus, a bacteria, a fungus, a protozoa or ametazoan.

In another preferred embodiment, at least one analyte is a nucleic acid.

In another preferred embodiment, the analyte(s) are not nucleic acids.

When the analyte or one of the analytes is a nucleic acid, it ispreferably a nucleic acid making it possible to diagnose an infection,for example an infection caused by a virus, a bacteria, a fungus, aprotozoa or a metazoan.

Typically, this may involve one or several antigens and/or one orseveral antibodies and/or one or several specific nucleic acids of:

-   -   a virus, such as HIV (Human Immunodeficiency Virus), in        particular HIV-1 or HIV-2, HBV (Hepatitis B Virus), HCV        (Hepatitis C Virus), HPV (Human Papilloma Virus), HTLV (Human        T-Lymphotropic Virus), in particular HTLV-I or HTLV-II,    -   a parasite, such as a parasite that may cause toxoplasmosis (in        particular Toxoplasma gondii), malaria (in particular a parasite        of the Plasmodium genus, for example Plasmodium falciparum,        Plasmodium vivax, Plasmodium ovale, Plasmodium malariae or        Plasmodium knowlesi) or Chagas disease (in particular        Trypanosoma cruzi) in humans or non-human animals, or    -   a bacteria, such as a bacteria able to cause syphilis (Treponema        pallidum) or Lyme disease (in particular a bacteria from the        Borrelia genus) in humans or non-human animals.

“Parasite” here refers to a metazoan or a protozoa acting as parasitewith respect to a body and causing parasitosis. A parasite within themeaning of the invention is therefore not a virus, a bacteria or afungus.

The analyte or at least one of the analytes may also be a marker fordisease, such as a marker of a cardiovascular disease or a diabetesmarker, a marker of the evolution of the disease, such as hepatitis, amarker of the evolution of an infection caused by a virus, a bacteria, afungus or a parasite, a marker of resistance to a treatment, for exampleto an antiviral treatment, an antibiotic treatment or a cancertreatment.

Several (for example, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, fifteen or more than fifteen)analytes as described in the present application may be detectedsimultaneously in a same sample or a same mixture of samples during amultiplex analysis method. This may make it possible to diagnose, in asame sample or a same mixture of samples, one or several infections ordiseases, the evolution of an infection or disease, a condition(pathological or not), a risk of developing a condition (pathological ornot) or a marker of resistance to a treatment in a subject.

The analytes detected during a multiplex analysis method may be of thesame nature (for example only antibodies, only antigens or only nucleicacids) or of different natures (for example, at least one antigen and atleast one antibody).

Capture Ligand

A capture ligand is a specific molecule of a compound of interest.

A capture ligand is preferably specific to an analyte to be detected ina sample. It generally involves an antibody, an antigen, a peptide, acarbohydrate, a lipid or a nucleic acid.

In one advantageous embodiment, the capture ligand is not a nucleicacid. The capture ligand(s) are for example selected from the groupconsisting of an antibody, an antigen, a peptide, a carbohydrate and alipid.

A capture ligand is fixed to the surface of a solid support at a spot.

In one preferred embodiment, the capture ligand(s) is (are) an antibody,an antigen or a nucleic acid.

In one more preferred embodiment, the capture ligand(s) are an antibodyor an antigen.

When the capture ligand, or one of the capture ligands, is an antibody,it for example involves a monoclonal antibody or a polyclonal antibody.

Detection Ligand

A detection ligand is a specific molecule of a compound of interest. Itin particular makes it possible to detect a compound of interest fixedto a capture ligand.

A detection ligand of an analyte is specific to an analyte to bedetected in a sample. It in particular makes it possible to detect ananalyte fixed to a capture ligand.

A detection ligand may be an antibody, an antigen, a peptide, acarbohydrate, a lipid or a nucleic acid. It is preferably an antibody oran antigen.

In another embodiment, the or one of the detection ligand(s) is (are) anucleic acid.

In another embodiment, the detection ligand is not a nucleic acid. Thedetection ligand(s) are for example selected from the group consistingof an antibody, an antigen, a peptide, a carbohydrate and a lipid.

When the detection ligand, or one of the detection ligands, is anantibody, it for example involves a monoclonal antibody or a polyclonalantibody.

The detection ligand or one of the detection ligands is preferably amarked detection ligand, i.e., a detection ligand to which a detectionmarker (which may for example be biotin or a peroxidase) is attached.The detection marker is attached (i.e., coupled) to the detection ligandcovalently or non-covalently, preferably covalently. The detectionligand or one of the detection ligands may be coupled to a direct orindirect detection marker.

When the detection ligand is not marked, its detection may be done byusing a specific marked antibody of said detection ligand.

A detection ligand may be identical to the used capture ligand or one ofthe used capture ligands, with the exception of any presence of adetection marker, and/or bind to the analyte to which it is specific atthe same zone as that bonded by the capture ligand or one of the captureligands, in particular when the analyte to which it is specific is inthe form of a complex having at least two identical bonding zones. Thus,when one of the analytes that one wishes to detect is an antibody, it ispossible to use, as capture ligand and detection ligand of said analyte,a same specific antigen of said antibody or two different antigensincluding at least one shared zone (at least one shared epitope)allowing the recognition by the bivalent antibody.

A capture ligand and a detection ligand may also be specific to separatezones at an analyte.

In one particular embodiment, a detection ligand and a capture ligand(for example, antibodies) specific to a same analyte (for example, anantigen) do not bond to the same location on said analyte. For example,the detection ligand can bond to a zone of said analyte to which it isspecific that is separated from the bonding zone of the capture ligand,for example so as to avoid competition of the capture ligand and thedetection ligand with respect to the compound to which they arespecific, due to a steric hindrance.

Detection Marker of the Detection Ligand

A detection marker coupled to the detection ligand may be a directmarker or an indirect marker.

A direct marker is a marker whose signal can be detected directly, i.e.,without requiring the prior addition of a reporter.

A direct marker is for example selected from the group consisting of aradioisotope, a fluorophore, a heavy element from the periodic tablesuch as a lanthanide, a luminescent compound, a transition metal such asruthenium, a chromogenic, and colored, fluorescent or luminescentnanoparticles.

A “luminescent” compound may be an electroluminescent compound, athermoluminescent compound or a chemiluminescent compound. In onepreferred embodiment, the luminescent compound is a chemiluminescentcompound.

One example luminescent compound (more specifically, thermoluminescentcompound) that may be used as a direct marker consists of silicananoparticles comprising (for example doped with) molecules of adioxetane compound, in particular the 1,2-dioxetane compound, or aderivative of a dioxetane compound, for example a derivative of1,2-dioxetane.

An indirect marker is a marker for which detection of the signalrequires the prior addition of a reporter (also called first reporter)and, if said reporter itself is coupled to an indirect detection marker,the addition of a second reporter of the indirect detection markercoupled to said first reporter.

A reporter is a substrate of an indirect marker or a moleculespecifically bonding to an indirect marker, said molecule itself being adirect or indirect marker or itself being coupled to a direct orindirect marker.

An indirect marker is for example selected from the group consisting ofan enzyme, a ligand of a ligand-receptor pair, a receptor of aligand-receptor pair, a hapten, an antigen and an antibody.

A ligand or a receptor of a ligand-receptor pair is for example biotin,an analogue of biotin, avidin, streptravidin, neutravidin ordigoxigenin.

A preferred indirect marker according to the invention is an enzyme,preferably an enzyme producing a luminescent compound by reaction with asubstrate.

The detection of the signal of an indirect marker requires the prioraddition of a reporter of said indirect marker.

A reporter of an enzyme is for example a substrate of said enzyme.

A reporter of the biotin is, for example, avidin, streptavidin orneutravidin, preferably coupled with a direct marker or an indirectmarker, such as an enzyme or a catalyst.

A reporter of the biotin is, preferably, coupled with a direct orindirect detection marker, such as an enzyme or a catalyst.

An example enzyme is peroxidase, for example horseradish peroxidase (HRPor POD), alkaline phosphatase or luciferase.

One preferred biotin reporter according to the invention is streptavidincoupled with a peroxidase, preferably horseradish peroxidase. Thedetection of the signal of the biotin then requires adding streptavidincoupled with the peroxidase, then adding the substrate of theperoxidase.

In this preferred embodiment, the detection ligand or one of thedetection ligands is coupled to a direct or indirect detection markerselected from among a peroxidase enzyme or a biotin.

When the analyte is a nucleic acid, the detection marker or one of thedetection markers may be detected by fluorescence, for example by usingdirect or indirect marking with a fluorophore, bioluminescence,preferably by chemiluminescence, for example by using direct markingwith a luminescent compound or indirect marking with an enzyme, inparticular a peroxidase, producing a luminescent compound by reactionwith a substrate.

When the analyte is a compound of the protein type, the detection markeror one of the detection markers is preferably detected by luminescence,for example by using direct or indirect marking with a compound of theprotein type, in particular a chemiluminescent compound or indirectmarking with an enzyme as reporter producing a luminescent compound byreaction with a substrate.

In one particular embodiment of the invention, the detection marker orone of the detection markers used has, as substrate, luminol,isoluminol, an acridine, coelenterazine, dioxetane or peroxyoxaliccompound, or one of their derivatives, and in particular a compounddescribed in the publication Dodeigne C. et al (2000), Talanta 51,415-439, “Chemiluminescence as diagnostic tool. A review”.

In one particular embodiment, the detection marker or one of thedetection markers used has, as substrate, luminol, an analogue ofluminol, for example isoluminol, or one of their derivatives.

“Luminol” refers to 3-aminophthalhydrazide, also called5-amino-2,3-dihydro-phthalazine-1,4-dione. The raw formula for luminolis C₈H₇N₃O₂. As an example, it is possible to use the ELISTAR ETA CUltra ELISA (Cyanagen, Italy) substrate described in the examples.

“Isouminol” Refers to 4-aminophthalhydrazide.

A derivative of luminol or an analogue of luminol is preferably amolecule obtained respectively from the luminol or the analogue of theluminol, through all possible modification(s) (for example, chemicaland/or enzymatic). A derivative of luminol or an analogue of luminol isfor example a substrate of a peroxidase enzyme, the reaction of saidperoxidase enzyme with a derivative of the luminol or the analogue ofthe luminol making it possible to produce a chemiluminescent compound.

A derivative of the isoluminol may for example be aminoethylisoluminol(or AEI), aminoethylethylisoluminol (or AEEI), aminobutylisoluminol (orABI), aminobutylethylisoluminol (or ABEI), aminopentylethylisoluminol(or APED, aminohexylisoluminol (or AHI), aminohexylethylisoluminol (orAHEI), aminooctylmethylisoluminol (or AOMI) or aminooctylethylisoluminol(or AOEI), as described in the publication Dodeigne C. et al (2000),Talanta 51, 415-439, “Chemiluminescence as diagnostic tool. A review”.

In one particular embodiment of the invention, the “luminol, analogue ofthe luminol (for example isoluminol), acridine, coelenterazine,dioxetane or peroxyoxalic compound, or one of their derivatives” may becontributed in the form of a composition (or formulation) comprisingsaid luminol, acridine, coelenterazine, dioxetane or peroxyoxaliccompound, or one of their derivatives.

Resistant Control Marker

The present invention is based on the use of one or several resistantcontrol markers (for example, a mixture of resistant control markers)making it possible to control the quality of spot(s) present on thesurface of a solid support, in particular the presence, location and/orintegrity of the spot(s) present on the surface of a solid support, thiscontrol (or one of these controls) being able to be done at the end ofthe analysis method carried out.

A resistant control marker is a compound able to be detected, forexample by producing a signal. The produced signal may for example be aradioactive signal or a light signal. It may also involve a controlmarker (for example, a colored control marker) that is detected by lightabsorption.

A light signal corresponds to the emission of a light, in particular ina given wavelength range.

A resistant control marker can for example be a marker emitting light byfluorescence, phosphorescent or luminescence, in particularelectroluminescence, thermoluminescence or chemiluminescence.

When the or one of the resistant control markers is a marker emittinglight by luminescence, it preferably emits light by chemiluminescence.

One preferred resistant control marker is a marker emitting light byfluorescence.

As its name indicates, a resistant control marker according to theinvention is resistant.

“Resistant control marker” here refers to a marker whereof a signal isdetected at the end of an analysis method. A resistant control marker isin particular a control marker that remains, in full or at leastpartially, fixed at a spot on the surface of a solid support, during thepreparation method of the solid support and also during the analysismethod implementing said support, and that is capable of producing asignal detectable at the end of the analysis method, i.e., including inthe presence of a detection marker of a detection ligand of an analyteand/or one or several reporters in the case of one or several indirectdetection markers.

In the expression “marker capable of producing a signal detectable atthe end of the analysis method”, “capable” means that the resistantcontrol marker present in a spot produces a signal detectable at thatspot at the end of the analysis method, when said analysis method hastaken place with no deterioration of said spot.

The expressions “able to be detected”, “whereof a signal is detected”,“capable of producing a detectable signal” or “producing a detectablesignal” at the end of the analysis mean, in particular in the case wherethe resistant control marker is a fluorophore, that the “detectedsignal” (the signal produced by the resistant control marker andmeasured on the imaging sensor at the corresponding pixel zone) minus(i.e., from which one subtracts) the mean signal of the surroundingpixels is around three standard deviations above the level of thesurrounding pixel signals. When the detected signal minus the meansignal of the surrounding pixels reaches at least three standarddeviations above the level of the signals of the surrounding pixels,said detected signal is qualified as “detectable signal”.

A control marker within the meaning of the invention is thus resistantto the washing and incubation steps with the different reagents usedduring an analysis method, such as the detection ligand(s), thereporter(s), the substrate(s), the sample, one or several diluents.

In particular, a resistant control marker according to the invention isresistant to the use of the detection marker(s) of a detection ligand ofan analyte, if applicable the (first) reporter of the detectionmarker(s) of a detection ligand of an analyte and if applicable the(second) reporter of the indirect marker coupled to said first reporter.

In one preferred embodiment, a resistant control marker according to theinvention is resistant to the use of one or several enzymes, inparticular an alkaline phosphatase and/or a peroxidase, and to thesubstrate(s) of said enzyme(s).

One preferred control marker according to the invention is resistant tothe use of a substrate selected from the group consisting of luminol,isoluminol or one of their derivatives.

In one advantageous embodiment, a resistant control marker according tothe invention is also resistant to the use of a composition (orformulation) comprising a substrate selected from the group consistingof luminol, isoluminol or one of their derivatives. Said composition mayfor example comprise hydrogen peroxide and/or chemical molecules (orcofactors) contributing to the efficacy of the luminol, isoluminol orone of their derivatives.

“Resistant to the use of a compound” or “resistant to a compound” heremeans that the control marker is resistant to the use of said compoundduring an analysis method. In particular, the control marker isresistant to being placed in the presence of said compound.

To determine whether a given compound is a resistant control markerwithin the meaning of the invention, it is possible to implement themethod comprising the following steps:

-   -   a) depositing said compound on the surface of a solid support,        to form at least one spot,    -   b) preferably, saturating the surface of the solid support,    -   c) preferably, drying the surface of the solid support,    -   d) optionally carrying out one or more washing steps,    -   e) optionally placing the spots in the presence of a detection        ligand,    -   f) optionally carrying out one or more washing steps,    -   g) optionally placing the spot(s) in the presence of at least        one reporter or at least one enzyme, for example a peroxidase        enzyme that may or may not be coupled to a reporter,    -   h) optionally carrying out one or more washing steps,    -   i) placing the spot(s) in the presence of at least one substrate        (said substrate preferably being that which will be used in the        analysis method in which one wishes to implement said resistant        control marker), preferably a substrate of a peroxidase enzyme,        for example luminol,    -   j) measuring the signal produced by said compound at the        spot(s), and    -   k) if the signal measured in step j) is a detectable signal,        concluding that said compound is a resistant control marker.

The “detectable signal” is in particular as defined above.

The type of signal measured in step j) depends on the tested compound.One skilled in the art knows what type of signal must be detected andhow to detect it, based on the tested compound.

The or one of the detection ligand(s) in step e) can be coupled with adetection marker of the biotin type, the or one of the reporters in stepg) can be a reporter of the streptavidin type coupled to an enzyme, forexample a peroxidase, and/or the or one of the substrates in step i) canbe a substrate of said enzyme, for example luminol, an analogue ofluminol, and/or a derivative of luminol or an analogue of luminol.

Surprisingly, the inventors have thus shown the resistant nature ofcontrol markers having simply been deposited on the surface of a solidsupport, mixed with a compound of interest, for example a captureligand, in order to form spots. Thus, in one particular embodiment ofthe invention, a resistant control marker according to the invention isnot fixed covalently to the surface of the solid support and/or is notcoupled covalently to a capture ligand.

Lastly, in one preferred embodiment of the invention, a resistantcontrol marker interferes little, or preferably not at all, with thesignal produced by a detection marker of a detection ligand of ananalyte used in an analysis method.

“Signal produced by the detection marker of a detection ligand of ananalyte” refers to the signal produced by the detection marker of thedetection ligand of an analyte itself or corresponding to the detectionmarker of the detection ligand. Indeed, for example, in the case of anenzyme or another type of indirect marker, the signal is not produced bythe detection marker of the detection ligand of an analyte itself, butby the reporter or a compound produced by the reaction of a reporter,for example an enzyme, with its substrate; in this case, this signalcorresponds to the detection marker of the detection ligand of ananalyte.

The expression “a resistant control marker that interferes little withthe signal produced by the detection marker of a detection ligand of ananalyte” means that the functionality of the analysis method for theuser is not affected by the presence of the resistant marker, inparticular that the decrease in sensitivity is less than 40%, preferablyless than 35%, more preferably less than 30%, still more preferably lessthan 25%. According to one particular embodiment, this expressionfurthermore means that there is no deterioration of the specificity.

The expression “there is no deterioration of the specificity” means thatthe increase in the threshold value is less than or equal to 40%,preferably less than or equal to 35%, more preferably less than or equalto 30%, and still more preferably less than or equal to 25%.

The threshold value is the average of the signals obtained for negativesamples at the end of the analysis method plus 12 times the standarddeviation of the signals of these samples.

A resistant control marker can be neutral, or positively or negativelycharged.

“Positively or negatively charged molecule” means, in the presentapplication, a molecule that includes a global charge that isrespectively positive or negative, in particular that globally includesone, two, three, four or more than four respectively positive ornegative charges.

A resistant control marker can comprise or be coupled to a carriermolecule, for example a protein such as BSA.

When the resistant control marker “comprises” a carrier molecule, itgenerally comprises or consists of a control marker (for example, afluorophore) and a carrier molecule (for example, BSA), said controlmarker being coupled to said carrier molecule. This coupling can make itpossible to make a control marker resistant that would not be resistantwithout coupling.

The coupling between a control marker or a resistant control marker anda carrier molecule results from a covalent or noncovalent bond betweenthe resistant control marker and the carrier molecule, preferably acovalent bond.

A carrier molecule able to be coupled covalently or non-covalently tothe control marker or the resistant control marker is for exampleselected from the group consisting of BSA, an immunoglobulin (alsocalled Ig), a dextran, polylysine and a mixed copolymer, in particular acopolymer of different amino acids (lysine-tyrosine copolymer, forexample).

Another example of a carrier molecule able to be coupled covalently ornon-covalently to the control marker or the resistant control marker isan oligonucleotide or a polynucleotide, in particular a DNA or RNA.

The resistant control marker according to the invention may for examplebe a marker selected at the end of the selection method for a resistantcontrol marker as defined below.

Resistant Control Fluorophore

One preferred resistant control marker according to the invention is afluorophore. In this case, it is called resistant control fluorophore.

A fluorophore, also called fluorochrome or fluorescent molecule, is asubstance, in particular a chemical or protein substance (or polypeptidesubstance), capable of emitting fluorescent light after excitation witha light energy.

A fluorophore generally comprises several conjugated aromatic coresand/or planar and cyclic molecules that have one or several bonds π.

A fluorophore may be a fluorescent protein, for example B-Phycoerythrin.

A resistant control marker according to the invention, in particular aresistant control fluorophore, is preferably characterized in that itsexcitation spectrum does not overlap the emission spectrum of adetection marker of a detection ligand of an analyte or corresponding toa detection marker of a detection ligand of an analyte (used in ananalysis method) and in that its emission spectrum does not overlap, orpartially overlaps, the emission spectrum of a detection marker of adetection ligand of an analyte or corresponding to a detection marker ofa detection ligand of an analyte (used in an analysis method).

The “emission spectrum or excitation spectrum corresponding to adetection marker of a detection ligand” is for example the emissionspectrum or the excitation spectrum of a direct marker or a reporter ofan indirect detection marker coupled to the detection ligand, a reporterof an indirect detection marker coupled to a reporter (in particular ofan indirect detection marker of a detection ligand), or a compoundproduced by the reaction of an enzyme-type reporter with its substrate.

In the case of a resistant fluorophore according to the invention, theexcitation spectrum may correspond to the absorption spectrum.

A preferred resistant control marker according to the invention, inparticular a preferred resistant control fluorophore, is a fluorophorecharacterized in that its excitation spectrum does not overlap theemission spectrum of the luminol, one of its analogues, in particularthe isoluminol, and/or one of their derivatives, and in that itsemission spectrum does not overlap or only partially overlaps theemission spectrum of the luminol, one of its analogues, in particularisoluminol, and/or one of their derivatives.

“Emission spectrum of the luminol, one of its analogues, in particularthe isoluminol, and/or one of their derivatives” refers to the emissionspectrum of the chemiluminescent compound resulting from the reaction ofthe luminol, one of its analogues, in particular isoluminol, or one oftheir derivatives with a peroxidase enzyme.

Preferably, a resistant control marker according to the invention, inparticular a resistant control fluorophore, is characterized in that itsexcitation spectrum does not overlap the emission spectrum of theluminol, the isoluminol or one of their derivatives.

When the emission spectrum of a resistant control marker according tothe invention, in particular of the resistant control fluorophore,partially overlaps the emission spectrum of the detection marker of oneor several detection ligands or corresponding to the detection marker ofone or several detection ligands, it is advantageous to use a filtermaking it possible to eliminate the wavelength emitted by said detectionmarker to keep only the specific signal emitted by the fluorophoreand/or a filter making it possible to eliminate the wavelengths emittedby said fluorophore, to keep only the specific signal emitted by saiddetection marker.

In one preferred embodiment, there is no energy transfer by resonancebetween:

-   -   the resistant control marker(s) according to the invention, in        particular the resistant control fluorophore(s), and    -   the detection marker(s) of a detection ligand and/or the        compound(s) producing a signal corresponding to the detection        marker(s) of a detection ligand used in the analysis method,        in particular between the resistant control marker(s) according        to the invention (in particular the resistant control        fluorophore(s)) and a luminescent compound obtained by reaction        of the luminol, one of its analogues, for example isoluminol,        and/or one of their derivatives with a peroxidase enzyme.

In one advantageous embodiment, the resistant control marker(s)according to the invention, in particular the resistant controlfluorophore(s), have excitation wavelength ranges and emissionwavelength ranges that are strictly greater than the emission wavelengthranges of the luminol, one of its analogues, for example isoluminol,and/or one of their derivatives, or strictly smaller than the emissionwavelength ranges of the luminol, one of its analogues, for exampleisoluminol, and/or one of their derivatives.

As an example, when luminol is used as substrate, a resistant controlmarker according to the invention, in particular a preferred resistantcontrol fluorophore according to the invention, does not emit lightaround 425 nm, and in particular 375 nm to 550 nm, preferably from 375nm to 580 nm, more preferably from 350 nm to 580 nm. In other words,when luminol is used as substrate, a preferred resistant controlfluorophore according to the invention emits light outside wavelengthsfrom 375 nm to 550 nm, from 375 nm to 580 nm or from 350 nm to 580 nm.It may for example emit light only at wavelengths less than (or lessthan or equal to) 375 nm, 370 nm, 360 nm or 350 nm, or only atwavelengths greater than (or greater than or equal to) 550 nm, 560 nm,570 nm, 580 nm, 590 nm or 600 nm.

The expression “from a value X to a value Y” means that the boundaries Xand Y are included.

In one particular embodiment, surprisingly, the basic pH of the reactionmedium containing the luminol, an analogue of luminol and/or one oftheir derivatives, the presence of peroxide, one or several agentsfavoring electron transfers and an acylation catalyst in the reactionmedium do not cause a spectral shift at the origin of a problem in thedetection of the signal emitted by the receiving control markeraccording to the invention, and in particular by the resistant controlfluorophore according to the invention.

According to one embodiment of the invention, a resistant control markeraccording to the invention, in particular a resistant controlfluorophore according to the invention, has an absorption window thatdoes not comprise the wavelength range corresponding to the signalemitted by the detection marker of the detection ligand and inparticular that does not comprise the emission wavelength range of theluminol. For example, one preferred resistant control fluorophoreaccording to the invention does not absorb wavelengths from 375 to 530nm, from 375 to 550 nm, or from 350 to 580 nm (inclusive). Thus, it isfor example possible to use a fluorophore that absorbs only wavelengthsless than (or less than or equal to) 375, 370, 360 or 350 nm, or afluorophore that absorbs only wavelengths greater than (or greater thanor equal to) 530, 540, 550, 560, 570, 580, 590 or 600 nm.

According to one particular embodiment, a resistant control marker maybe a neutral fluorophore, a positively charged fluorophore or anegatively charged fluorophore or a fluorescent protein, saidfluorophore optionally being coupled to a carrier molecule, for examplea protein such as BSA.

In one preferred embodiment, the resistant control fluorophore comprisesat least one positively charged functional group, and in particular atleast one amine function.

According to one particular embodiment, a resistant control markercomprises or consists of at least one fluorophore and at least onecarrier molecule (for example, BSA), said at least one fluorophore beingcoupled to said at least one carrier molecule, and said at least onefluorophore for example being able to be a neutral, positively chargedor negatively charged fluorophore, or a fluorescent protein.

A carrier molecule is in particular as defined above in the “analyte”paragraph.

A resistant control fluorophore is, for example, selected from the groupconsisting of a coumarin, a rhodamine, a carbopyronine, an oxazine,benzopyrylium, a phycoerythrin and derivatives thereof. Said fluorophoreis optionally coupled to a carrier molecule, for example a protein suchas BSA.

A resistant control fluorophore is, for example, selected from the groupconsisting of a coumarin, a rhodamine, a carbopyronine, an oxazine, abenzopyrylium derivative, their derivatives, and a phycoerythrin. Aresistant control fluorophore is for example chosen from among thecompounds described in application WO00/64986 A1 and/or marketed by thecompany Atto-Tec.

One preferred resistant control fluorophore is, for example, selectedfrom the group consisting of a carbopyronine, an oxazine, an oxazinederivative, a benzopyrylium derivative, and a phycoerythrin.

Another preferred resistant control fluorophore is selected from thegroup consisting of a carbopyronine derivative, an oxazine, an oxazinederivative, a benzopyrylium derivative, and a phycoerythrin.

One still more preferred resistant control fluorophore is, for example,selected from the group consisting of a carbopyronine, a benzopyryliumderivative, and a phycoerythrin.

Another still more preferred resistant control fluorophore is, forexample, selected from the group consisting of a carbopyroninederivative, a benzopyrylium derivative, and a phycoerythrin.

One example includes the Atto 633 carbopyronine marketed by the companyAtto-Tec and its derivatives, in particular an amine derivative, as wellas derivatives of benzopyrylium like those marketed by the companyDyomics, in particular Dye 634 or Dye 630 when they are coupled to acarrier molecule, for example BSA, or the amine derivative of Dye 634 orDye 630, coupled or not coupled to a carrier, for example BSA.

One preferred carbopyronine according to the invention is a moleculecomprising the following basic structure:

A carbopyronine able to be used as resistant control fluorophore, likethe amine derivative from Atto 633, for example has the followingcharacteristics: maximum absorption wavelength=629 nm, molar absorptioncoefficient at the maximum absorption wavelength=1.3×10⁵ M⁻¹ cm⁻¹,maximum emission wavelength=657 nm and quantum efficiency=64%.

A benzopyrylium derivative able to be used as resistant controlfluorophore for example has the following characteristics:

-   -   maximum absorption wavelength (in ethanol): 635 nm,    -   maximum emission wavelength (in ethanol): 658 nm, and    -   molar absorption coefficient at the maximum absorption        wavelength: 200,000 M⁻¹ cm⁻¹.

This for example involves the fluorophore called Dye 634 (in its formcoupled to a carrier molecule, for example BSA) with formula:

Dye 634 can also be used as resistant control fluorophore in its amineform (Dye 634-amine). It may be used coupled or not coupled to a carriermolecule, and in particular BSA.

Still another benzopyrylium derivative able to be used as resistantcontrol fluorophore for example has the following characteristics:

-   -   maximum absorption wavelength (in ethanol): 636 nm,    -   maximum emission wavelength (in ethanol): 657 nm, and    -   molar absorption coefficient at the maximum absorption        wavelength: 200,000 M⁻¹ cm⁻¹.

This for example involves Dye 630 (in its form coupled to a carriermolecule, for example BSA).

Dye 630 has the following formula:

Dye 630 can also be used in its amine form (Dye 630-amine), coupled ornot coupled to a carrier molecule, for example BSA.

Enzyme as Resistant Control Marker

Another example of a resistant control marker according to the inventionis an enzyme for example producing a luminescent compound by reactionwith a substrate of said enzyme.

Preferably, the luminescent compound is a chemiluminescent compound.

An enzyme able to be used as resistant control marker according to theinvention is for example an alkaline phosphatase or a luciferase.

An alkaline phosphatase substrate is for example the Lumi-Phos 530 orLumi-Phos substrate more marketed by Lumigen.

A luciferase substrate is for example the luciferin or coelenterazinesubstrate.

When the resistant control marker is an enzyme producing a luminescentcompound, the detected signal corresponding to the resistant controlmarker is the signal emitted by the luminescent compound produced by thereaction of said enzyme with a substrate of said enzyme.

Detection of the Signal

The signal produced by the detection marker(s) of the detection ligandor produced by the resistant control marker(s) is detected directly orindirectly.

Depending on the marker used, the signal can for example be detected byfluorescence or luminescence, in particular by chemiluminescence.

The signal emitted by a marker of the fluorophore type can be readdirectly by fluorescence.

A marker of the enzyme type requires the addition of a substrate makingit possible to produce a detectable product, for example the addition ofa substrate allowing the production of light.

A resistant control marker emitting light by electroluminescencerequires the addition of a Ruthenium complex and the addition ofTripropylamine (TPA), as well as the application of an electric currentallowing light production.

As indicated above, an indirect marker of the biotin type requires theaddition of a reporter, preferably a reporter coupled to a direct orindirect detection marker.

If the reporter is coupled to an indirect marker of the enzyme type, forexample peroxidase, it is necessary to add, in a later step, thesubstrate of that enzyme, for example the luminol or an analogue of theluminol, such as isoluminol or a derivative of the luminol or ananalogue of the luminol.

The signal may advantageously be detected using a device according tothe invention, as defined below.

In another preferred embodiment, the signal emitted by the resistantcontrol marker(s) is detected by fluorescence and the signal emitted bythe detection marker(s) of the detection ligand is emitted byluminescence, for example by chemiluminescence.

Generally, the chemiluminescence reaction is done using a kit comprisingat least two solutions.

The first solution comprises the substrate for the peroxidase, forexample the luminol, the isoluminol and/or a derivative of the luminolor the isoluminol, and an electron mediator; the second solutioncomprises an oxidizer. As an example, it is possible to use thefollowing kits: “Immun-star western C” (Bio-Rad, United States),“ELISTAR ETA C Ultra ELISA” (Cyanagen, Italy), “Supersignal West Pico”(Thermo Scientific, United States), “Chemiluminescent Sensitive PlusHRP” (Surmodics, United States).

Solid Support for Improved Detection of at Least One Analyte in a Sample

The support(s) used to carry out the analysis method according to theinvention are solid supports.

According to one particular embodiment, a solid support is obtainedusing the method for preparing a solid support according to theinvention.

A solid support can be made from any material appropriate to carry outthe analysis method. A solid support is for example a support with abase of a polymer or a mixture of polymers.

An appropriate solid support is for example a support made frompolystyrene, polypropylene, poly(meth)acrylate, polybutadiene orcombinations thereof.

Another example of an appropriate solid support is a membrane, forexample a membrane made from nitrocellulose, PVDF (polyvinylidenefluoride), nylon or combinations thereof.

Still another example of an appropriate solid support is an inorganicsupport, for example a glass slide and/or a metal support.

One preferred solid support is made from polystyrene or polypropylene.

A solid support comprises at least one compartment (also called analysiszone), preferably at least two compartments.

According to one particular embodiment of the invention, a solid supportcomprises a single compartment. Said single compartment may be acompartment including one or several walls. Alternatively, said singlecompartment can have no walls and then be comparable to the solidsupport itself. The bottom of the compartment can then consist of theupper face of the solid support. One example of such a solid supportcomprising a single compartment that may or may not include one orseveral walls is a slide or a membrane.

According to one particular embodiment of the invention, the solidsupport, which may for example be a microplate or a membrane, comprisesat least two compartments.

When the solid support comprises at least two compartments, they areisolated from one another, such that they do not communicate with oneanother, i.e., such that the various compositions or solutions used forthe analysis cannot circulate from one compartment to another during theanalysis. Thus, a solution added into one compartment will not go intothe other compartments. For example, the compartment(s) comprise or aremade up of a bottom and one or several walls, said wall(s) isolating thecompartment(s) from one another such that they do not communicate withone another.

Typically, at least one (for example one or two) compartment of thesolid support is used per sample to be analyzed.

In one particular embodiment of the invention where the solid support(for example a slide or a membrane) comprises a single compartment,typically, at least one (for example one or two) solid support is usedper sample to be analyzed.

The solid support is for example a microplate. In this case, one examplecompartment is a well.

A microplate is typically a microplate with 96 wells or 384 wells.

The present invention thus relates to a solid support for detecting atleast one analyte in at least one sample, characterized in that saidsolid support comprises at least one compartment comprising at least onespot, said spot comprising at least one resistant control marker and atleast one capture ligand.

A resistant control marker is in particular as defined above, inparticular in the paragraph “resistant control marker”, “resistantcontrol fluorophore” and “enzyme as resistant control marker”, or asobtained using the selection method defined below in the paragraph“selection method of a resistant control marker”.

In particular, said resistant control marker(s) are markers that remainat least partially fixed at said spot on the surface of the solidsupport during the implementation of a method for detecting at least oneanalyte, so as to produce a detectable signal at the end of thedetection method.

A compartment of the solid support intended to analyze a samplecomprises at least one spot, for example two spots, three spots, fourspots or five spots, or at least six spots, preferably six spots, sevenspots, eight spots, more preferably at least nine spots, for examplenine spots, ten spots, eleven spots, twelve spots, thirteen spots,fourteen spots, fifteen spots, sixteen spots or more than sixteen spots.

“Spot” here refers to a zone on the surface of a compartment of a solidsupport comprising at least one resistant control marker and/or at leastone compound of interest, preferably at least one resistant controlmarker and at least one compound of interest, for example a captureligand. The resistant control marker(s) and the compound(s) of interestthus fix at the same time to said zone on the surface of thecompartment, through noncovalent physicochemical interactions (inparticular of the weak bond type, for example, ionic, van der Waals,hydrogen and/or hydrophobic) and/or by covalent bonds.

A spot may comprise, aside from the compound(s) of interest, at leastone polymer, in particular at least one polymer including hydrophilicgroups, for example at least one hydrogel.

In one particular embodiment, all of the spots of a compartment,preferably all of the spots of a solid support, comprise a singleresistant control marker or a single mixture of resistant controlmarkers that may be used at a same concentration in all of the spots orat different concentrations.

Alternatively, different (at least two) resistant control markers and/ordifferent (at least two) mixtures of resistant control markers can beused in spots of a same compartment of a solid support.

The present invention particularly relates to a solid support fordetecting at least one analyte in at least one sample, characterized inthat said solid support comprises at least one compartment comprising atleast one spot, said spot comprising a single resistant control markerand at least one capture ligand.

Preferably, all of the spots intended to detect an analyte, inparticular all of the spots comprising a capture ligand, comprise onlyone resistant control marker, which is preferably the same in all of thespots intended to detect an analyte of a same compartment, and morepreferably, which is the same in all of the spots intended to detect ananalyte of all of the compartments of a solid support.

A spot corresponds to a well-defined zone, for example comprised between0.0078 mm² to 5.309 mm², preferably from 0.196 mm² to 3.142 mm², morepreferably comprised from 0.503 mm² to 2.011 mm².

A spot may have a discoid, cylindrical or hemispherical shape, orapproximately discoid, cylindrical or hemispherical shape, for exampleoval, in particular when the solid support is a microplate or a slide.

Alternatively, a spot may have a square or rectangular shape (inparticular a strip), for example when the solid support is a membrane,or any other shape.

The spots are obtained using techniques well known by those skilled inthe art, such as those disclosed in U.S. Pat. Nos. 7,470,547 B2,6,576,295 B2, 5,916,524 A and 5,743,960 A.

For example, a spot is obtained by depositing at least one drop of asolution containing a determined quantity of at least one resistantcontrol marker and at least one compound of interest in a specificlocation on the surface of the compartment of the solid support.

When a spot comprises at least one polymer (for example at least onehydrogel), said spot may be obtained by depositing at least one drop ofa solution containing a determined quantity of at least one resistantcontrol marker and at least one compound of interest in a specificlocation on the surface of the compartment on which said polymer hasbeen previously deposited.

The surface of a compartment is also called “solid phase”.

A compound of interest is generally a capture ligand, in particular acapture ligand of an analyte. One or several spots of a compartment canserve as control spot and thus comprise a capture ligand that is notintended to detect an analyte of the sample, or comprise anothercompound of interest, or not comprise any compound of interest.

The present invention particularly relates to a solid support fordetecting at least one analyte in a sample, characterized in that saidsolid support comprises at least two compartments comprising at leastone spot, said spot comprising at least one resistant control marker andat least one capture ligand.

In one particular embodiment, some or all of the compartments of a solidsupport have the same spot composition.

In another particular embodiment, some or all of a solid support orcompartments of a solid support comprises or consists of several (forexample two) distinct groups (or types) of spots or compartments, eachof the separate groups having a different spot composition (due to thenumber of spots and/or the resistant control marker(s) and/or thecapture ligand(s) and/or the compound(s) of interest present in thespots of each group).

Preparation of an Analysis Support Comprising a Resistant Control Marker

The present invention also relates to a method for preparing a solidsupport for detecting at least one analyte in at least one samplecomprising the following steps:

-   -   a) depositing, on the surface of at least one compartment,        preferably at least two compartments, of a solid support, a        mixture comprising at least one resistant control marker and at        least one capture ligand, to obtain a spot,    -   b) repeating step a) n−1 times, n being an integer greater than        or equal to 1, to obtain n spots intended to detect an analyte        on the surface of said compartment(s),    -   c) optionally, saturating the surface of said compartment(s),        and    -   d) optionally, drying the surface of said compartment(s).

The solid support, the resistant control marker(s) and the captureligand(s) are in particular as defined above.

In step a), a mixture comprising at least one resistant control markerand at least one capture ligand is deposited on the surface of at leastone compartment of a solid support, preferably at least two compartmentsof a solid support, to obtain a spot.

In step a), said resistant control marker(s) are markers that remain atleast partially fixed at said spot on the surface of the solid supportduring the implementation of a method for detecting at least oneanalyte, so as to produce a detectable signal at the end of thedetection method.

A resistant control marker is in particular as defined in the presentapplication.

Said resistant control marker(s) are preferably fluorophores, forexample a mixture of fluorophores. The fluorophore(s) can be fluorescentchemical molecules or fluorescent proteins.

Said capture ligand(s) are preferably selected from the group consistingof an antibody, an antigen, a nucleic acid and combinations thereof.

In another preferred embodiment, said capture ligand(s) are not nucleicacids.

For example, said capture ligand(s) are selected from the groupconsisting of an antibody and an antigen.

The mixture is preferably a solution.

A resistant control marker is present in the mixture at a concentrationthat does not interfere with the fixing of the capture ligand(s) to thesurface of the compartment and allows detection of the signal producedby said resistant control marker at the end of the analysis method.

As an example, the mixture comprises 0.1 to 100 μg/ml of a captureligand and 0.01 to 100 μg/ml of a control marker, preferably in a buffersolution, for example a TBS (Tris Buffered Saline) solution.

The concentration of a resistant control marker in the mixture can forexample be determined using the method comprising the following steps:

-   -   depositing at least two solutions on the surface of a solid        support, each solution comprising said resistant control marker        and a capture ligand, the concentration of the resistant control        marker increasing from one solution to the other and the lowest        concentration of the resistant control marker being greater than        or equal to a predefined minimum concentration,    -   optionally, saturating the surface of the solid support, i.e.,        placing the surface of the solid support in the presence of an        agent making it possible to avoid nonspecific bonds to the solid        support,    -   optionally, drying the surface of the solid support,    -   placing a sample including an analyte having a specific capture        ligand in the presence of spots,    -   placing a specific detection ligand of said analyte in the        presence of spots, said detection ligand being coupled to a        direct or indirect detection marker,    -   when said detection ligand is coupled to an indirect detection        marker, adding a reporter (also called first reporter) of said        indirect detection marker and when said (first) reporter is        coupled to an indirect detection marker, adding a second        reporter of the indirect detection marker coupled to said        (first) reporter of the indirect detection method of the        detection ligand,    -   detecting a signal produced by the resistant control marker,    -   detecting a signal produced by the detection marker of the        detection ligand, and    -   selecting a concentration of resistant control marker that makes        it possible to detect both the signal produced by the resistant        control marker and the signal produced by the detection marker        of the detection ligand.

The concentration of resistant control marker preferably makes itpossible to detect both the signal produced by the resistant controlmarker and the signal produced by the detection marker of the detectionligand, without significant loss of sensitivity relative to a spot notcomprising the resistant control marker, i.e., the functionality of theanalysis method for the user is not affected by the presence of theresistant marker, in particular that the decrease in sensitivity is lessthan 40%, preferably less than 35%, more preferably less than 30%, stillmore preferably less than 25%.

According to one particular embodiment, the concentration of resistantcontrol marker makes it possible to detect both the signal produced bythe resistant control marker and the signal produced by the detectionmarker of the detection ligand without deterioration of the specificity.

The expression “there is no deterioration of the specificity” means thatthe increase in the threshold value is less than or equal to 40%,preferably less than or equal to 35%, more preferably less than or equalto 30%, and still more preferably less than or equal to 25%.

The threshold value is the average of the signals obtained for negativesamples at the end of the analysis method plus 12 times the standarddeviation of the signals of these samples.

When a detection ligand is coupled to an indirect detection marker, areporter of said indirect detection marker is therefore added. When saidreporter (called first reporter) is itself is coupled to an indirectdetection marker, for example an enzyme, a second reporter, for examplea substrate, of the indirect detection marker coupled to said firstreporter is added.

The predefined minimum concentration of resistant control marker can forexample be determined using the following method:

-   -   depositing at least two solutions on the surface of a solid        support, each solution comprising a resistant control marker and        not comprising capture ligand, the concentration of the        resistant control marker increasing from one solution to the        other, to form at least two spots,    -   optionally, saturating the surface of the solid support, i.e.,        placing the surface of the solid support in the presence of an        agent making it possible to avoid nonspecific bonds to the solid        support,    -   optionally, drying the surface of the solid support,    -   optionally carrying out at least one of the following steps:        -   (i) performing one or more washing steps,        -   (ii) placing the spot(s) in the presence of a detection            ligand,        -   (iii) placing the spot(s) in the presence of a reporter,        -   (iv) placing the spot(s) in the presence of a substrate,    -   detecting the signal produced by the resistant control marker,        and    -   selecting the minimum concentration of resistant control marker        that allows the detection of a signal.

The mixture (or solution) can be deposited on the surface of the solidsupport or a compartment of the solid support manually, but preferablyis done automatically by an appropriate device.

Thus, as previously indicated, a resistant control marker fixes on thesolid phase of the analysis support at the same time as the captureligand(s) present in the mixture (or solution).

In one particular embodiment of the invention, each spot of acompartment of the solid support intended to detect at least one analytecomprises at least one different capture ligand, preferably intended todetect an analyte by spot. However, several spots of a compartment maycomprise at least one same capture ligand.

In one particular embodiment of the invention, a same spot may compriseseveral different capture ligands (for example, several antibodiesand/or antigens), which are generally specific to a same pathology,infection or disease to be detected (in particular specific to a samevirus, a same bacteria, a same fungus or a same parasite), the evolutionof an infection or disease, a condition (pathological or not) of thesubject, a risk of developing a condition (pathological or not) or amarker of resistance to a treatment.

Several spots or all of the spots of a compartment may comprise captureligands intended to detect a same analyte; this for example involvesdifferent specific capture ligands of a same analyte or even a samecapture ligand present at different concentrations in the spots.

In one particular embodiment, the resistant control marker or themixture of resistant control markers is the same in all of the spots,and it may or may not be added at a same concentration in each spot.

Alternatively, different resistant control markers (at least two) and/ordifferent mixtures of resistant control markers can be used in differentspots of a compartment.

A compartment may also comprise one or several spots with no resistantcontrol marker. However, preferably, all of the spots of saidcompartment comprise a resistant control marker.

A compartment may also comprise one or several spots with no captureligand, but preferably comprising another compound of interest.

When a compartment comprises one or several spots with no captureligand, the spot(s) preferably comprise a resistant control marker.

In one particular embodiment of the invention, all of the compartmentsof the support have the same spot composition.

In another particular embodiment of the invention, some or all of thecompartments of a solid support comprise or consist of several (forexample two) distinct groups of compartments, each of the distinctgroups having a different spot composition.

Steps a) and b) are carried out in some or all of the compartments ofthe solid support, preferably, in all of the compartments of the solidsupport.

The mixture deposited on the surface of at least one compartment of thesolid support is incubated for example from several seconds to severalhours, for example at a temperature comprised from 4° C. to 40° C.

The method comprises a potential step c) for saturating the surface ofsaid compartment(s) of the solid support, i.e., testing of placing thesurface of the solid support in the presence of an agent making itpossible to avoid nonspecific bonds to the solid support. The saturationstep is in particular intended to prevent the nonspecific fixing ofcompounds, during the implementation of the analysis method.

The agent making it possible to avoid nonspecific bonds to the solidsupport is for example a saturation solution well known by those skilledin the art.

In the potential step d), the surface of the compartment(s) is dried.

The drying is for example done at 56° C. or 37° C. or at ambienttemperature.

In one particular embodiment, step a), step b) and/or step c) arefollowed by one or several washing steps.

The present invention also relates to a method for preparing a solidsupport as defined above, comprising a subsequent step e) consisting ofchecking the quality of the spots.

If the quality check of a spot is positive, said spot can be used aspart of an analysis method.

If the quality check of a spot is negative, said spot cannot be used aspart of an analysis method, potentially as well as the compartmentcontaining said spot.

The present invention also relates to a kit, characterized in that itcomprises or consists of at least one solid support according to theinvention or obtained using the preparation method according to theinvention and, if applicable, at least one composition or solution to beused to carry out an analysis method according to the invention and/oruser instructions.

Device for Improved Detection (or Device for Double Detection)

The present invention also aims to provide a device for detecting atleast one analyte in a sample, said device comprising:

-   -   means for detecting a first signal and a second signal produced        at a solid support, and    -   means for defining a reading grid from the location of said        first signal and for reading said second signal at said reading        grid.

According to one particular embodiment, this reading corresponds to aquantification of the second signal at the regions of interest.

The detection of the first signal can be done before, after orsimultaneously with the detection of the second signal.

The solid support is in particular as defined above.

The first signal can be a fluorescence or luminescence signal, forexample by chemiluminescence.

The second signal can be a fluorescence or luminescence signal, forexample by chemiluminescence.

In one preferred embodiment, the first signal is a fluorescence signaland the second signal is a luminescence signal, preferably bychemiluminescence (or vice versa).

The means for detecting a first signal and a second signal produced at asolid support are called optical bench.

An optical bench may for example comprise or be made up of:

-   -   a lighting system,    -   a telecentric objective,    -   a filter wheel, said telecentric objective preferably being        coupled on its output lens to said filter wheel, and    -   a camera.

The lighting system is preferably a steerable lighting system.

The lighting system makes it possible to illuminate the solid supportmore or less intensity with, for example to reveal the fluorescence orthe geometry of the solid support used.

The telecentric objective is preferably large. In particular, thetelecentric objective covers the entire solid support.

The telecentric objective makes it possible to image the entire surfaceto be measured without deforming the image, to eliminate the parallaxerror inherent to standard objectives and to guarantee signalhomogeneity over the entire viewed surface.

The telecentric objective is preferably coupled on its output lens to afilter wheel.

The filter wheel makes it possible to present different filters betweenthe telecentric objective and the camera (also called image acquisitioncamera). In one preferred embodiment, it is thus possible to select thewavelengths reaching the sensor of the camera to be able, in one case,to separate the excitation signals of the lighting from those emitted byat least one resistant control marker (in particular at least onefluorophore) through the presence of a filter, and, in the other case,to collect the maximum amount of signal produced by at least onedetection marker of a detection ligand of an analyte through thepresence of a neutral window not filtering the signal but guaranteeingthe maintenance of the optical properties of the assembly.

The camera makes it possible to acquire an image with variable exposuretimes.

The optical bench thus defined makes it possible to take a series ofimages of the same support, with different lighting, filtering and/orimage acquisition parameters, such as:

-   -   an image in the visible domain (also called positioning image),        for example by using the lighting from the lighting system, but        no specific filter,    -   a fluorescence image (also called detection image), by using the        lighting from the lighting system and a specific filter for the        sought fluorescence,    -   an image with no lighting and no specific filter (also called        analysis image), for example to reveal a chemiluminescence        signal.

The means for defining a reading grid from the location of said firstsignal and for reading the second signal at said reading grid cancomprise or be made up of an imaging system.

In one advantageous embodiment, the optical bench is thereforeassociated with an imaging system making it possible to carry out ananalysis method comprising several steps that in particular guaranteethe accuracy and robustness of the analysis performed, said stepscomprising:

-   -   1. searching for and positioning the solid support and the        compartment(s) of the solid support, in particular owing to the        positioning image; the purpose of this processing is to reduce        any mechanical positioning errors of the solid support and make        it possible to use mechanics not having a high positioning        precision;    -   2. from the position of the compartment(s) determined in step 1,        positioning, in each compartment, a theoretical grid        representing the theoretical position of each of the spots; this        positioning can be done from a reference grid, for example        described in the analysis system, said reference grid indicating        the coordinates of each of the spots relative to a reference        point of the compartment; the reference point of the compartment        is for example the center of the compartment;    -   3. searching, using the detection image done with the lighting        and the fluorescence filter, for all of the fluorescent events        existing in the compartment(s) of the solid support, and        optionally, rejecting certain events using a selection of events        by their size and shape so as to eliminate those that are        completely outside the expected specifications;    -   4. comparison between:        -   the position of the events previously detected and            considered valid,        -   the theoretical position of the expected spots as defined in            step 2;    -   5. for each of the expected spots, association with the closest        detected event and intersecting the surface of the theoretical        spot so as to form “theoretical spot”/“fluorescent event” pairs        making it possible to detect the missing spots. For each        theoretical spot, a distinct and unique fluorescent event must        exist;    -   6. characterization of each of the detected spots using        diameter, shape, distance from the theoretical position criteria        making it possible to guarantee the integrity of the detected        spots;    -   7. for each of the detected spots, definition of the        corresponding region of interest, called reading grid, in        particular defined by the surface and coordinates of the        detected spots; and    -   8. reading of the second signal on the reading grid defined in        step 7, in particular quantification of the second signal on the        analysis image at this region of interest, i.e., the reading        grid.

Analysis Method with Quality Check of the Spots

The analysis method according to the invention allows the detection ofat least one analyte in at least one sample.

The present invention in particular relates to a method for detecting atleast one analyte in at least one sample comprising the following steps:

-   -   a) placing a sample to be analyzed in the presence of the        spot(s) of the compartment of a solid support, said spot or at        least one of said spots comprising at least one resistant        control marker and at least one capture ligand of an analyte,    -   b) placing at least one detection ligand of an analyte in the        presence of the spot(s) of said compartment, said detection        ligand of an analyte being coupled to a direct or indirect        detection marker,    -   c) when at least one detection ligand of an analyte is coupled        to an indirect detection marker, placing a reporter of said        indirect detection marker in the presence of the spot(s) of said        compartment,    -   d) when the reporter used in step c) is coupled to an indirect        detection marker, placing a reporter of the indirect detection        marker coupled to said reporter used in step c) in the presence        of the spot(s) of said compartment,    -   e) detecting a signal produced by at least one resistant control        marker in said compartment,    -   f) defining a reading grid from the location of the signal        detected in step e),    -   g) optionally, detecting a signal produced by at least one        detection marker of a detection ligand of an analyte, and    -   h) optionally, reading the signal detected in step g) on the        reading grid defined in step f).

As defined above, the or said resistant control markers are capable ofproducing a detectable signal at the end of an analysis method, i.e., toproduce a detectable signal at a spot when the analysis method has takenplace without deterioration of said spot. In particular, the or saidresistant control markers are capable of producing a detectable signalduring step e) of the method above.

The present invention in particular relates to a method for detecting atleast one analyte in at least one sample comprising the following steps:

-   -   a) placing a sample to be analyzed in the presence of the        spot(s) of the compartment of a solid support, said spot or at        least one of said spots comprising at least one resistant        control marker and at least one capture ligand of an analyte,    -   b) placing at least one detection ligand of an analyte in the        presence of the spot(s) of said compartment, said detection        ligand of an analyte being coupled to a direct or indirect        detection marker,    -   c) when at least one detection ligand of an analyte is coupled        to an indirect detection marker, placing a reporter of said        indirect detection marker in the presence of the spot(s) of said        compartment,    -   d) when the reporter used in step c) is coupled to an indirect        detection marker, placing a reporter of the indirect detection        marker coupled to said reporter used in step c) in the presence        of the spot(s) of said compartment,    -   e) detecting a signal produced by at least one resistant control        marker in said compartment,    -   f) defining a reading grid from the location of the signal        detected in step e),    -   g) optionally, detecting a signal produced by at least one        detection marker of a detection ligand of an analyte, and    -   h) optionally, reading the signal detected in step g) on the        reading grid defined in step f),    -   said resistant control marker(s) being markers that remain at        least partially fixed at the spot on the surface of the solid        support during the implementation of said method for detecting        at least one analyte, so as to produce a detectable signal        during step e).

Steps a) to d) are still done before steps e) to h).

Step g) can be carried out before, after or at the same time as step e).In one particular embodiment, step g) is carried out before or afterstep e).

Step f) is still carried out after step e).

Step f) is still carried out after step e) and before step h), and canbe carried out before, after or at the same time as step g).

Step h) is still carried out after steps e) to g).

Steps a) to f) and, if applicable, step g) and/or step h) are done foreach compartment of a solid support comprising at least one spotcomprising at least one resistant control marker and at least onecapture ligand of an analyte, in which a sample is analyzed.

Steps g) and/or h) may not be done if the reading grid defined in stepf) does not indicate any zone for reading the signal to be detected instep g).

If step h) is carried out, step g) is also carried out.

The method may advantageously comprise one or several washing steps, forexample between each or some of steps a) to d).

The washing of each compartment intended to analyze a sample comprisesat least one cycle, preferably 3 to 6 cycles, for distributing andaspirating a volume (for example 400 μL) of a washing solution (forexample, a Tris NaCl 0.01 M buffer solution, pH 7.4, doped with Tween 20at 0.1%).

According to one particular embodiment of the invention, no operationand in particular no pipetting, distributing, agitating, aspirating orwashing step is done between steps e) and g), irrespective of the orderin which steps e), f) and g) are carried out.

The expression “place a compound X in the presence of the spots of acompartment” means that the compound X is added into a compartmentcomprising said spots, said compartment being intended to analyze asample.

When at least two compounds are to be placed in the presence of thespot(s) of the compartment during a same step and/or when at least twosteps a) to d) are done at the same time, said compounds may be placedin the presence of said spot(s) separately, i.e., contributed in theform of separate compositions; alternatively, said compounds or some ofthe compounds may be placed in the presence of the spot(s) of acompartment in the form of one or several mixtures.

The detection method is in particular carried out using a solid supportas defined above or obtained by the preparation method as defined above.

The resistant control marker(s), the spot(s), the capture ligand(s) ofan analyte are in particular as defined above.

In particular, the resistant control marker(s) are markers that remainat least partially fixed at said spot on the surface of the solidsupport during the implementation of said method for detecting at leastone analyte, so as to produce a detectable signal during step e), i.e.,when the analysis method has taken place without deterioration of aspot.

The present invention particularly relates to a method as defined above,characterized in that said or one of said resistant control markers is(are) one (or several) fluorophore(s), for example one or severalfluorescent chemical molecules or one or several fluorescent proteins,for example a mixture of fluorophores.

The present invention particularly relates to a method as defined above,characterized in that said or one of said resistant control markers is aresistant control marker, in particular a fluorophore, whereof theexcitation spectrum does not overlap the emission spectrum of the signalemitted by or corresponding to the detection marker of said detectionligand(s) of an analyte and whereof the emission spectrum does notoverlap, or partially overlaps, the emission spectrum of orcorresponding to the detection marker of said detection ligand(s) of ananalyte.

The present invention for example relates to a method as defined above,characterized in that said or one of said resistant control markers is aresistant control marker, in particular a fluorophore, whereof theexcitation spectrum does not overlap the emission spectrum of the signalemitted by a luminescent compound obtained by reaction of the luminoland/or an analog and/or a derivative of the luminol or an analogue ofthe luminol with a peroxidase enzyme and whereof the emission spectrumdoes not overlap, or partially overlaps, the emission spectrum of aluminescent compound obtained by reaction of the luminol and/or ananalogue and/or a derivative of the luminol or an analogue of theluminol with a peroxidase enzyme.

In one preferred embodiment, the detection method as defined abovecomprises the following steps:

-   -   a) placing a sample to be analyzed in the presence of the        spot(s) of the compartment of a solid support, said spot or at        least one of said spots comprising at least one resistant        control marker and at least one capture ligand of an analyte,    -   b) placing at least one detection ligand of an analyte in the        presence of the spot(s) of said compartment, said detection        ligand of an analyte being coupled to an indirect detection        marker,    -   c) placing a reporter of said indirect detection marker in the        presence of the spot(s) of said compartment,    -   d) when the reporter used in step c) is coupled to an indirect        detection marker, placing a reporter of the indirect detection        marker coupled to said reporter used in step c) in the presence        of the spot(s) of said compartment,    -   e) detecting a signal produced by at least one resistant control        marker in said compartment,    -   f) defining a reading grid from the location of the signal        detected in step e),    -   g) optionally, detecting a signal produced by at least one        detection marker of a detection ligand of an analyte, and    -   h) optionally, reading the signal detected in step g) on the        reading grid defined in step f).

The present invention particularly relates to a method as defined above,characterized in that:

-   -   the signal produced by at least one detection marker of a        detection ligand of an analyte is the light emitted by a        chemiluminescent compound obtained by reaction of a peroxidase        with the luminol, an analogue of the luminol and/or a derivative        of the luminol or an analogue of the luminol, and    -   the signal produced by the or said resistant control marker(s)        is a light emitted outside the wavelengths from 375 to 550 nm,        from 375 to 580 nm, or from 350 to 580 nm (inclusive).

Preferably, the resistant control marker(s) have an excitation spectrumand, preferably, an emission spectrum outside the wavelengths from 375to 550 nm, from 375 to 580 nm, or from 350 to 580 nm (inclusive).

The analysis method is for example an immunoassay, and in particular animmuno-enzymatic assay, such as an ELISA (Enzyme-Linked ImmunoSorbentAssay) test. The analysis method can also be used to detect nucleicacids.

In step a), a sample to be analyzed is placed in the presence of thespot(s) of a compartment of the solid support.

When several samples are analyzed and the solid support comprisesseveral compartments, step a) comprises placing a sample in the presenceof the spot(s) of at least one compartment of a solid support, saidsolid support comprising at least as many compartments comprising atleast one spot comprising at least one resistant control marker and atleast one capture ligand of an analyte as the number of samples to beanalyzed or several solid supports are used.

When several samples are analyzed and the solid support comprises asingle compartment, step a) comprises adding a sample into thecompartment of a solid support (or, when said compartment is comparableto the solid support itself, adding a sample on said solid support), andone uses at least as many solid supports comprising a compartmentcomprising at least one spot comprising at least one resistant controlmarker and at least one capture ligand of an analyte as the number ofsamples to be analyzed.

When several samples are analyzed, a different compartment is used foreach sample. It is also possible to use several compartments of one orseveral solid supports to analyze a same sample, in particular if saidcompartments have a different spot composition.

Steps a) and b) can be done at the same time, step a) before step b), orstep b) before step a). When step b) is done before step a), the methoddoes not comprise a washing step between these two steps a) and b).

When they are present, steps c) and d) are preferably done after stepsa) and b) and there is preferably at least one washing step betweensteps a) and b) and steps c) and d).

When they are present, steps c) and d) can be done at the same time, orstep c) before step d). When step d) is done before step c), the methoddoes not comprise a washing step between these two steps d) and c).

When it is present, step d) is preferably done after step c) and thereis at least one washing step between steps c) and d).

In step b), at least one detection ligand of an analyte is placed in thepresence of the spot(s) of said compartment.

Step b) is in particular done in each compartment intended to analyze asample.

The detection ligand(s) of an analyte are in particular as definedabove.

Preferably, step b) comprises adding at least one detection ligand ofeach analyte to be detected. When different detection ligands of ananalyte are used, they may be added at the same time or one after theother, all or part of the ligands being able to be contributed in theform of separate compositions or of one or several mixtures. Some of thedetection ligands of an analyte may be added simultaneously with stepa), preferably followed by a washing step before adding the rest of thedetection ligands of an analyte.

When detection ligands of an analyte are added successively during stepb), each addition of at least one analyte detection ligand can befollowed by a washing step of the compartment intended to analyze asample.

The performance of steps c) and d) depends on the detection marker ofthe detection ligand and, if applicable, the detection marker of thereporter of the detection marker of the detection ligand.

Thus, step c) is carried out when at least one detection ligand of ananalyte is coupled to an indirect detection marker.

Preferably, the detection ligand(s) of an analyte are coupled to thesame detection marker. If at least two detection ligands of an analyteare coupled to different indirect detection markers, step c) is carriedout for each indirect detection marker, in order to detect the signal ofeach marker.

Step d) is carried out when at least one reporter (also called firstreporter) used in step c) is coupled to an indirect detection marker.The reporter used in step d) is called second reporter.

When step d) is carried out, step c) is therefore also carried out.

In step e), a signal produced by at least one resistant control markeris detected in said compartment.

If at least two resistant control markers are used, step e) preferablycomprises detecting a signal produced by each of said resistant controlmarkers.

The detection of the signal produced by at least one resistant controlmarker in particular makes it possible to localize each spot comprisingat least one resistant control marker in each compartment of the solidsupport.

The signal produced by at least one resistant control marker ispreferably a signal emitted by fluorescence.

In step f), a reading grid is defined from the location of the signaldetected in step e).

The reading grid indicates precisely in which zone(s) of the compartmentthe second signal must be read (or “analyzed” or “taken into account” or“interpreted”). In particular, a second signal detected in step g)outside the or one of the zones defined in the reading grid is not takeninto account in the reading (or the “analysis” or the “interpretation”)of the second signal during step h).

It is particularly advantageous to define said zones precisely accordingto the contour of each spot.

Step f) for defining the reading grid therefore in particular comprisesverifying the quality of the spots, from the signal detected in step e),which corresponds to at least one resistant control marker.

“Quality of the spots” refers to the presence, location and/or integrityof the spot(s).

The integrity of the spots comprises the size and shape of the spot.

A quality check of a spot is in particular positive if the spot ispresent in the compartment in a position in line with the expectedposition, if it has a well-defined contour, if its shape is in line withthe acceptability criteria, for example, if it has a discoid,approximately discoid, for example oval shape or a shape with acircularity greater than 80%, and if it does not intersect another spotof the compartment.

A “circularity greater than 80%” means that the detected spot has asufficient circularity to guarantee that it has not been damaged by allof the treatments done during the analysis method.

For example, a quality check of a spot is positive if a signal isdetected in step e), this signal has a well-defined contour, a discoidshape, an approximately discoid shape, for example oval or a with acircularity greater than 80%, and if it does not intersect the signalproduced by another spot of the compartment.

If no signal is detected in step e) at a spot or if the spot does notmeet the quality control for at least one other of the reasons indicatedabove, the second signal optionally detected in step g) at said spot isnot taken into consideration when reading the second signal in step h),potentially as well as the second signal detected at each spot of thecompartment corresponding to said spot.

In steps e) and g), the detection of a signal preferably comprises theacquisition of a signal.

The use of one or several resistant control marker(s) according to theinvention thus makes it possible to perform a reading of the secondsignal in the precise locations where the spots are located, making itpossible to improve the sensitivity of the analysis, while securing therendered results, in particular by making it possible to eliminate falsepositives or false negatives related to a spot flaw.

The present invention particularly relates to the method as definedabove, characterized in that the signal produced by at least onedetection marker of a detection ligand of an analyte is a luminescentsignal, in particular a chemiluminescent signal, for example the lightemitted by reaction of the luminol and/or an analogue and/or aderivative of the luminol or of an analogue of the luminol with aperoxidase enzyme.

The present invention more particularly relates to a method as definedabove, characterized in that the signal produced by at least oneresistant control marker is detected by fluorescence and the signalproduced by at least one detection marker of a detection ligand of ananalyte is detected by luminescence, in particular chemiluminescence.

In one preferred embodiment, at least steps e) to h) are carried outusing a same device, for example the device as described in the “devicefor improved detection” section.

In another preferred embodiment, all of the steps of the method arecarried out using a same device, for example the device as described inthe “device for improved detection” section.

Method for Selecting a Resistant Control Marker

The present invention also relates to a method for selecting a resistantcontrol marker, comprising the following steps:

-   -   a) depositing a marker to be tested on the surface of a solid        support, to form at least one spot,    -   b) optionally, saturating the surface of the solid support,    -   c) optionally, drying the surface of the solid support,    -   d) carrying out at least one of the following steps:        -   (i) performing one or more washing steps,        -   (ii) placing the spot(s) in the presence of a detection            ligand,        -   (iii) placing the spot(s) in the presence of a reporter,        -   (iv) placing the spot(s) in the presence of a substrate,    -   e) selecting a marker that produces a signal at the end of step        d).

The marker to be tested is a compound that produces (for example, emits)a signal able to be detected. For example, the marker to be tested is afluorophore or a luminescent compound.

The solid support is in particular as defined above.

Step a) comprises or consists of depositing a marker to be tested on thesurface of a solid support, to form at least one spot.

The marker can be deposited manually or automatically using anappropriate device.

The marker to be tested is generally deposited in the form of a solutioncomprising said marker.

Step a) can comprise depositing a same marker to be tested in severalspots, and increasing concentrations.

The solution deposited in step a) may or may not comprise a captureligand.

The method comprises a potential step b) for saturating the surface ofthe solid support. The saturation step is in particular intended toprevent the nonspecific fixing of compounds, during the implementationof the analysis method.

In step c), the surface of the solid support is optionally dried.

Generally, step a) and/or step b) are followed by one or several washingsteps a′) and/or b′).

Step d) comprises or consists of carrying out at least one of thefollowing steps, preferably at least two of the following steps, morepreferably at least three of the following steps:

-   -   (i) performing one or more washing steps,    -   (ii) placing the spot(s) in the presence of at least one        detection ligand,    -   (iii) placing the spot(s) in the presence of at least one        reporter, and/or    -   (iv) placing the spot(s) in the presence of at least one        substrate.

In one preferred embodiment, step d) comprises or consists of step (iii)and/or step (iv), preferably step (iii) and step (iv).

In another preferred embodiment, step d) comprises or consists of step(i), step (iii) and/or step (iv), preferably step (i), step (iii) andstep (iv).

In one preferred embodiment, step d) comprises at least step (i), step(ii), step (iii) and step (iv).

Steps (i) to (iv) can be done in any desired order.

However, in one preferred embodiment, step d) comprises the followingsteps and in the following order: step (ii), step (i), step (iii), step(i), step (iv) and step (i).

In another preferred embodiment, step d) comprises the following stepsand in the following order: step (ii), step (i), step (iii), step (i)and step (iv).

In another preferred embodiment, when these steps are present, step (ii)is carried out before step (iii) and/or before step (iv) and step (iii)is carried out before step (iv). Step (i) can be carried out betweensteps (ii) and (iii), (ii) and (iv) (in particular when step (iii) isabsent) and/or (iii) and (iv).

Preferably, the or one of the reporters of step (iii) is the reporter ofa detection marker coupled to at least one detection ligand of step (ii)and/or the or one of the substrates of step (iv) is a reporter of adetection marker coupled to a reporter.

For example, the or one of the detection ligand(s) of step (ii) iscoupled with a detection marker of the biotin type, the or one of thereporters of step (iii) is a reporter of the streptavidin type coupledto an enzyme, for example a peroxidase, and/or the or one of thesubstrates in step (iv) is a substrate of said enzyme, for exampleluminol, an analogue of luminol, and/or a derivative of luminol or ananalogue of luminol.

The selection method comprises a step e) for selecting a marker thatproduces a signal at the end of step d). In particular, the markerselected in step e) produces a signal at the end of step d). A“detectable signal” is in particular as defined above.

Preferably, step e) further comprises selecting a marker that does notinterfere or interferes little with the detection of the signal producedby a detection marker of a detection ligand.

The selection method according to the invention may also comprise:

-   -   optionally, first:        -   steps a) to c) above, in which the spot(s) formed in step a)            do not comprise a capture ligand,        -   optionally, a first preselection step comprising selecting a            marker that produces a detectable signal at the end of step            c),        -   optionally, steps d) to e) on the same support, preferably            said steps only being carried out with said marker selected            in the first preselection step, followed by a second            preselection step comprising selecting a marker that            produces a detectable signal at the end of step c),    -   secondly, steps a) to e) above, in which the spot(s) formed in        step a) comprises a capture ligand, steps d) to e) preferably        being done only with a marker selected in said first        preselection step and/or selected in said second preselection        step.

Use of a Resistant Control Marker

The present invention also relates to the use of at least one resistantcontrol marker in at least one spot intended to detect an analyte tosecure a method for detecting at least one analyte in a sample or atleast one sample.

“To secure a method for detecting at least one analyte in a sample or atleast one sample” here means guaranteeing the reliability of the resultsobtained at the end of said detection method, in particular by avoidingthe presence of “false negatives” or “false positives”.

A “false negative” is a negative result reflecting the absence of one orseveral analytes to be detected in a sample, whereas said analyte(s)were present in the sample and should have been detected.

A “false positive” is a positive result reflecting the presence of oneor several analytes to be detected in a sample, whereas said analyte(s)were absent from the sample.

The securing of the method for detecting at least one analyte in asample is in particular obtained by checking, at the end of thedetection method, the quality of the spot(s) intended to detect ananalyte and/or by improving the sensitivity of the detection of theanalytes, through the use of at least one resistant control marker.

The present invention thus relates to the use of at least one resistantcontrol marker in at least one spot intended to detect an analyte tosecure a method for detecting at least one analyte in a sample or atleast one sample, characterized in that it comprises:

-   -   checking the quality of said spot, after said spot has been        placed in the presence of the sample and at least one detection        ligand of an analyte to be detected, in particular at the end of        the analysis method, and/or    -   the reading of the signal produced by at least one detection        marker of a detection ligand of an analyte to be detected on a        reading grid defined from the location of the signal produced by        said resistant control marker(s).

The reading of the signal produced by at least one detection marker of adetection ligand of an analyte to be detected is therefore done on areading grid defined from the location of the signal produced by saidresistant control marker(s) and detected at the end of the analysismethod.

The present invention in particular relates to the use of at least oneresistant control marker in at least one spot intended to detect ananalyte to secure a method for detecting at least one analyte in asample or at least one sample, characterized in that it comprises:

-   -   checking the quality of said spot, after said spot has been        placed in the presence of the sample and at least one detection        ligand of an analyte to be detected coupled with an indirect        detection marker, a reporter (also called first reporter) of        said indirect detection marker, optionally a second reporter of        an indirect detection marker coupled to said first reporter,        and/or    -   the reading of the signal produced by at least one detection        marker of a detection ligand of an analyte to be detected on a        reading grid defined from the location of the signal produced by        said resistant control marker(s).

The present invention particularly relates to the use as defined above,characterized in that the quality control of the spot comprises orconsists of checking the presence, location and/or integrity of thespot.

The reading grid indicates the zone(s) of the compartment in which thesignal produced by the detection marker(s) of a detection ligand of ananalyte must be read.

Other features and advantages of the invention will better emergethrough the following examples, provided as an illustration andnon-limitingly.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Use of different fluorophores and resulting fluorescence imagesin different steps of the protocol described in example 1.

FIG. 2: Acquisition of the fluorescence signal of 12 wells comprising 9spots at the end of an analysis method according to the invention.

FIG. 3: Fluorescence image: Comparison of the theoretical reading grid(solid white circles) relative to the actual position of the spots byfluorescence (dotted white circles) based on the signal produced by theresistant control marker.

FIG. 4: Chemiluminescence image: Improvement of the accuracy of theanalysis method by defining the reading grid on the actual position ofthe spots detected through the resistant control marker (dotted line),versus the theoretical position grid (solid lines).

FIG. 5: Chemiluminescence image: Improvement of the accuracy of theanalysis method by verifying the integrity of the spots detected beforevalidating the results.

FIG. 6: Diagram of an optical bench comprising a lighting system, atelecentric objective, a filter wheel, said telecentric objective beingcoupled on its output lens to the filter wheel, and a camera.

EXAMPLES Example 1: Example Method for Selecting a Resistant ControlMarker

Materials and Method

Within each well of a polystyrene microplate (Greiner, Germany) aredeposited, well by well, drops of 500 nl of a fluorophore solution inthe buffer traditionally used for spotting of antigens or antibodies.The following fluorophores are used in this example: Atto 633-carboxylicacid (i.e., Atto 633-COOH or Atto 633) (supplier: ATTO-TEC; Germany),Atto 633-amine (i.e., Serive amine from Atto 633) (supplier: ATTO-TEC;Germany), Dye 634-carboxylic acid (i.e., Dye 634-COOH or Dye 634)(supplier: Dyomics, Germany), Dye 634-amine (i.e., Serive amine from Dye634) (supplier: Dyomics, Germany), Dye 630-amine (i.e., Serive aminefrom Dye 630) (supplier: Dyomics, Germany), APC (Allophycocyanin)(supplier Febico; Taiwan), B-Phycoerythrin (Febico; Taiwan). The bottomof each well of these microplates has molecule adsorption capacitiesknown in themselves by those skilled in the art. The surface of eachwell thus obtained is saturated with a saturation solution known initself by those skilled in the art; the wells are filled with thesaturation solution, the saturation solution is removed and the wellsare next dried; after a rehydration step of these wells, a substratesolution containing the luminol (ELISTAR ETA C Ultra ELISA (Cyanagen,Italy) (cf. example 2) is then added at a rate of 50 μL/well. Thefluorescence images are done at the different steps of the protocoldescribed above (i.e., after the steps for depositing drops, saturation,drying, rehydration, and after adding the substrate solution containingthe luminol), by using the Chemidoc™ MP System (Bio-Rad) having theappropriate filters for fluorophores.

Results

FIG. 1 describes the use of different fluorophores and resulting imagesin different steps of the protocol described above.

The fluorescence obtained with the 4 fluorophores is fully visible atthe end of the deposition of the drops on the plate. This fluorescencepersists for the Atto 633-amine, the allophycocyanin and theB-Phycoerythrin after elimination of the saturation solution (cf. spotsafter saturation). It also persists after adding the substrate solutioncontaining the luminol for the Atto 633-amine and for theB-Phycoerythrin, very weakly for the allophycocyanin. Similar resultsare obtained with the Dye 634-amine and the Dye 630-amine (results notshown). Conversely, no residual fluorescence is visible at the end ofthe washing step with the Dye 634-carboxylic acid and a fortiori afteradding the substrate solution, as well as for the Atto 633-carboxylicacid (results not shown).

Thus, the Atto 633-amine, the Dye 634-amine, the Dye 630-amine and theB-Phycoerythrin are resistant control markers according to the presentinvention.

Furthermore, as illustrated below in example 2, the Dye 634 coupled tothe BSA is also a resistant control marker according to the invention.

Example 2: Absence of Impact of the Presence of a Resistant ControlFluorophore on the Detection of an Analyte of Interest

Materials and Method

(i) Preparation of the microplate

Within each well of a polystyrene microplate (Greiner, Germany), aspotter robot is used to deposit 50 nL drops of a solution containingthe capture ligand(s) as well as a fluorophore selected using the methoddescribed in the application in rows.

The capture ligand solution can be:

-   -   either an antigen or mixture of antigens able to be made up of a        recombinant protein associated with one or more synthetic        peptides as part of an antibody detection test,    -   or an antibody or mixture of antibodies against the sought        marker in the case of an antigen detection test.

These capture ligand solutions contain the selected fluorophore, forexample the Atto 633-amine (Atto-tec, Germany) or a Dye 634-BSA complex,obtained using a protocol known by those skilled in the art from Dye 634(Dyomics, Germany) in NHS-ester (N-Hydroxysuccinimide ester) formcoupled with the BSA; these fluorophores are added at the appropriatedose, determined for each one; for the Dye 634-BSA, the indicated dosecorresponds to the concentration of the Dye 634-BSA complex. The bottomof each well of these microplates has adsorption capacities for thesedifferent proteins known in themselves by those skilled in the art.

The spots thus obtained are saturated with a saturation solution knownin itself by those skilled in the art. The plates are next dried.

(ii) Implementation of the Analysis Method

Description of the various elements used during the implementation ofthe analysis method:

Reporter

-   -   The Streptavidin-POD (S-POD) reporter is streptavidin (Roche,        Germany) coupled with Peroxidase (Roche Germany) according to        the method described by Nakane and Kawaoi [J Histochem        Cytochem (1974) Vol. 22, No. 12. pp. 1084-1091] known in itself        by those skilled in the art.

Wash Solution

-   -   Tris 10 mM buffer solution, pH 7.4, containing: NaCl 218 mM,        Tween20™ (trademark of the company Sigma) at 0.1%, Proclin300™        (trademark of the company Supelco) at 0.002%.

Developing Substrate

-   -   The ELISTAR ETA C Ultra ELISA developing substrate (Cyanagen,        Italy) is made up of two solutions: XLSE024L Luminol enhancer        solution (A) and XLSE024P Peroxide solution (B).        Description of the different steps carried out:        The test protocol comprises the following steps:

Step 1:

1. In each well of a microplate (comprising the spots) are distributed:

-   -   40 μl of sample: the sample can for example be a serum or a        control sample    -   40 μl of diluent        2. The mixture is incubated for 40 minutes at 37° C. with        agitation.        3. Three successive washes with at least 300 μl of wash solution        are done.        4. Next, an incubation step is done in the presence of the        detection ligand, then washing under the same conditions as        point 3.        5. Then, an incubation step of the reporter, then washing.        6. Lastly, a final developing step, including the addition of 25        μl of each of developing substrate solutions B and A.        7. The mixture is incubated for 1 minute at 37° C. with        agitation.        8. The readings are done with a Chemidoc™ reader to measure the        fluorescence and a Qview™ reader to measure the        chemiluminescence. The results of the readings are processed        directly by an image analysis system and recorded in Relative        Light Units (RLU); also, Relative Fluorescence Intensity (RFI).

Results

Each datum corresponds to an average of a triplicate; the fluorescencevalues correspond to the fluorescence signal level after deducting thebackground noise.

Results obtained with the Atto 633-amine fluorophore added into thespecific antibody solution of the marker of interest to be detected.

TABLE 1 Impact of the presence of the Atto 633-amine fluorophore on thedetection of the analyte for different fluorophore doses. E1, E2 and E3are positive samples, containing the antigen to be detected, atdifferent positivity levels. Atto 633-amine fluorophore Comparison ofthe Chemiluminescence chemiluminescence Composition of the signal signalrelative to the Sample deposit solutions RLU absence of fluorophore E1Antibody without fluorophore 1489.5 reference Antibody + Fluo 0.1 μg/ml1726  16% Antibody + Fluo 0.35 μg/ml 1703  14% Antibody + Fluo 1.2 μg/ml1377  −8% Antibody + Fluo 2.5 μg/ml 1438  −3% Antibody + Fluo 5 μg/ml1210 −19% E2 Antibody without fluorophore 4651 reference Antibody + Fluo0.1 μg/ml 5033  8% Antibody + Fluo 0.35 μg/ml 4861  5% Antibody + Fluo1.2 μg/ml 3679 −21% Antibody + Fluo 2.5 μg/ml 4115 −12% Antibody + Fluo5 μg/ml 3779 −19% E3 Antibody without fluorophore 2188 referenceAntibody + Fluo 0.1 μg/ml 2117  −3% Antibody + Fluo 0.35 μg/ml 2264  3%Antibody + Fluo 1.2 μg/ml 1650 −25% Antibody + Fluo 2.5 μg/ml 1479 −32%Antibody + Fluo 5 μg/ml 1226 −44%

The results shown in table 1 show the absence of impact of the presenceof the Atto 633-amine fluorophore on the detection of the analyte forfluorophore doses of 0.35 μg/ml or less. The calculation of thedetection limit of the analyte shows the absence of impact of thepresence of the fluorophore up to a dose of 1.2 μg/ml. The presence ofthe capture ligands only very slightly modifies the fluorescence (cf.table 2), which remains very significant and fully detectable at the endof the analysis.

TABLE 2 Impact of the presence of capture ligands on the fluorescencesignal (case of the Atto 633-amine fluorophore). E1 and E3 are positivesamples, containing the antigen to be detected, at different positivitylevels. N1 is a negative sample, not containing the antigen to bedetected. ‘Fluo’ stands for Fluorophore. Atto 633-NH2 fluorophoreComparison of the Fluorescence Chemiluminescence fluorescence signalComposition of the signal signal relative to the absence Sample depositsolutions RLU RLU of markers of interest E1 Fluorophore alone 0.1 μg/ml26843 54 −21% Antibody + Fluo 0.1 μg/ml 21158 1726 E3 Fluorophore alone0.1 μg/ml 29039 56 −31% Antibody + Fluo 0.1 μg/ml 20021 2117 N1Fluorophore alone 0.1 μg/ml 24999 57 −29% Antibody + Fluo 0.1 μg/ml17641 49Results obtained with the Dye 634-BSA fluorophore added into thespecific antibody solution of the marker of interest to be detected:

The results shown in table 3 show the absence of impact of the presenceof the Atto 634-BSA on the detection of the analyte for fluorophoredoses of 6 μg/ml or less, this dose being compatible with the detectionof the fluorescence at the end of analysis and the implementation of thedata processing method as described in the present application.

TABLE 3 Impact of the presence of the Atto Dye 634-BSA fluorophore onthe detection of the analyte for different fluorophore doses. E1, E2 andE3 are positive samples, containing the antigen to be detected, atdifferent positivity levels. ‘Fluo’ stands for Fluorophore. Dye 634-BSAfluorophore Comparison of the Chemiluminescence chemiluminescenceComposition of the signal signal relative to the Sample depositsolutions RLU absence of fluorophore E1 Antibody without fluorophore1856 reference Antibody + Fluo 3 μg/ml 1716  −8% Antibody + Fluo 6 μg/ml1782  −4% Antibody + Fluo 12 μg/ml 1562 −16% Antibody + Fluo 25 μg/ml1379 −26% Antibody + Fluo 50 μg/ml 1297 −30% E2 Antibody withoutfluorophore 5576 reference Antibody + Fluo 3 μg/ml 5471  −2% Antibody +Fluo 6 μg/ml 4908 −12% Antibody + Fluo 12 μg/ml 4133 −26% Antibody +Fluo 25 μg/ml 4231 −24% Antibody + Fluo 50 μg/ml 4629 −17% E3 Antibodywithout fluorophore 2183 reference Antibody + Fluo 3 μg/ml 2397  10%Antibody + Fluo 6 μg/ml 2184  0% Antibody + Fluo 12 μg/ml 1714 −21%Antibody + Fluo 25 μg/ml 1445 −34% Antibody + Fluo 50 μg/ml 1347 −38%Results obtained with the Atto 633-amine fluorophore added into theantibody solution corresponding to the antibodies to be detected:

The results shown in table 4 show the absence of impact of the presenceof the Atto 633-amine fluorophore on the detection of the analyte forthe fluorophore dose of 0.1 μg/ml or less. The presence of captureligands (cf. table 5) modifies the fluorescence, but the signal remainsvery significant, fully detectable at the end of analysis and usable toimplement the data processing method as described in the presentapplication.

TABLE 4 Impact of the presence of the Atto 633-amine fluorophore on thedetection of the analyte for different fluorophore doses. Samples S1 andS2 are positive samples, containing antibodies reacting selectively andrespectively relative to 2 types of antigens used in mixture:recombinant protein or synthetic peptide. ‘Fluo’ stands for Fluorophore.Atto 633-amine fluorophore Comparison of the Chemiluminescencechemiluminescence Composition of the signal signal relative to theSample deposit solutions RLU absence of fluorophore S2 Antigens withoutfluorophore 410 reference Antibody + Fluo 0.1 μg/ml 355 −13% Antigens +Fluo 0.35 μg/ml 351 −14% Antigens + Fluo 1.2 μg/ml 366 −11% Antigens +Fluo 2.5 μg/ml 330 −20% Antigens + Fluo 5 μg/ml 247 −40% S1 Antigenswithout fluorophore 1638 reference Antibody + Fluo 0.1 μg/ml 1547 −6%Antigens + Fluo 0.35 μg/ml 1215 −26% Antigens + Fluo 1.2 μg/ml 1261 −23%Antigens + Fluo 2.5 μg/ml 1253 −24% Antigens + Fluo 5 μg/ml 1194 −27%

TABLE 5 Impact of the presence of capture ligands on the fluorescencesignal (case of the Atto 633-amine fluorophore). Samples S1 and S2 arepositive samples, containing antibodies reacting selectively andrespectively relative to 2 types of antigens used: recombinant proteinor synthetic peptide. N2 is a negative sample, not containing antibodiesrecognizing the antigen to be detected. ‘Fluo’ stands for Fluorophore.Atto 633-amine fluorophore Comparison of the fluorescenceChemiluminescence fluorescence signal Composition of the signal signalrelative to the absence Sample deposit solutions RLU RLU of markers ofinterest N2 Fluorophore alone 0.1 μg/ml 70881 70 −47% Antigens + Fluo0.1 μg/ml 37781 67 S2 Fluorophore alone 0.1 μg/ml 72852 93 −45%Antigens + Fluo 0.1 μg/ml 39877 355 S1 Fluorophore alone 0.1 μg/ml 73721117 −47% Antigens + Fluo 0.1 μg/ml 39096 1547Results obtained with the Dye 634-BSA fluorophore added into theantibody solution corresponding to the antibodies to be detected:

TABLE 6 Impact of the presence of the Atto Dye 634-BSA fluorophore onthe detection of the analyte for different fluorophore doses. The sampleS1 is a sample containing antibodies reacting with respect to therecombinant protein used. ‘Fluo’ stands for Fluorophore. Dye 634-BSAfluorophore Comparison of the Chemiluminescence chemiluminescenceComposition of the signal signal relative to the Sample depositsolutions RLU absence of fluorophore S1 Antigens without fluorophore1066 reference Antigens + Fluo 3 μg/ml 1218 14% Antigens + Fluo 6 μg/ml1003 −6% Antigens + Fluo 12 μg/ml 1277 20% Antigens + Fluo 25 μg/ml 129121% Antigens + Fluo 50 μg/ml 1247 17%

The results shown in table 6 show the absence of impact of the presenceof the Dye 634-BSA fluorophore on the detection of the analyteirrespective of the fluorophore dose. The fluorescence signal obtainedat the end of analysis is fully detectable and usable to implement thedata processing method as described in the present application.

Example 3: Use of a Resistant Control Marker to Secure a Method forDetecting at Least One Analyte in a Sample

Materials and Method

(i) Preparation of the Microplate

Within each well of a polystyrene microplate (Greiner, Germany), aspotter robot is used to deposit 50 nL drops of a solution containingthe capture ligand(s) as well as a fluorophore selected using the methoddescribed in the application in rows.

The capture ligand solution can be:

-   -   either an antigen or mixture of antigens able to be made up of a        recombinant protein associated with one or more synthetic        peptides as part of an antibody detection test,    -   or an antibody or mixture of antibodies against the sought        marker in the case of an antigen detection test.

These capture ligand solutions contain the selected Atto 633-aminefluorophore (Atto-tec, Germany) at the appropriate dose comprisedbetween 0.1 to 0.5 μg/mL. The bottom of each well of these microplateshas adsorption capacities for these different proteins known inthemselves by those skilled in the art.

The spots thus obtained are saturated with a saturation solution knownin itself by those skilled in the art. The plates are next dried.

(ii) Implementation of the Analysis Method

Description of the various elements used during the implementation ofthe analysis method:

I. Reporter

The Streptavidin-POD (S-POD) reporter is streptavidin (Roche, Germany)coupled with Peroxidase (Roche Germany) according to the methoddescribed by P. Nakane and A. Kawaoi [J Histochem Cytochem (1974) Vol.22, No. 12. pp. 1084-1091] known in itself by those skilled in the art.

II. Diluents

a) Diluent Step 1

Tris buffer solution 50 mM, pH 7.5, containing: NaCl 150 mM, EDTA 20 mM,mouse IgG (Meridian) at 500 μg/mL, Cow's milk (100% skim) at 15%, Sheepserum at 10%, NaN3 at 0.095%.

b) Diluent of Conjugates 1

Tris buffer solution 50 mM, pH 7.5, containing: NaCl 150 mM; EDTA 20 mM,Chaps 0.1%, Glycerol 10%, NaN3 at 0.095%.

c) Diluent of Conjugates 2

Citrate buffer solution 50 mM, pH 6.7, containing: NaCl 150 mM, EDTA 5.6mM, Triton at 2%, Sheep serum at 10%, mouse IgG 500 μg/mL, Proclin300™(trademark of the company Supelco) at 0.5%, cow's milk (100% skim) at15%, Glycerol 10%. NaN3 at 0.095%.

d) Diluent of the Streptavidin-POD Reporter

Citrate buffer solution 50 mM, pH 6.7, containing: NaCl 2053 mM,Tween20™ (trademark of the company Sigma) at 0.5%, Proclin300™(trademark of the company Supelco) at 0.5%, cow's milk (100% skim) at7%, Glycerol 20%.

e) Wash Solution

Tris 10 mM buffer solution, pH 7.4, containing: NaCl 218 mM, Tween20™(trademark of the company Sigma) at 0.1%, Proclin300™ (trademark of thecompany Supelco) at 0.002%.

f) Developing Substrate

The ELISTAR ETA C Ultra ELISA developing substrate (Cyanagen, Italy) ismade up of two solutions: XLSE024L Luminol enhancer solution (A) andXLSE024P Peroxide solution (B).

III. Reaction Dishes

The immunological reactions take place in 96-well microplates (Greiner,Germany) made from polystyrene having a maximum volume of 392 μL perwell.

IV. Samples

The negative samples (serum or plasma) used come from the French bloodagency in Lille.

V. Optical Bench

The optical bench used is made up of the following elements:

-   -   a lighting system emitting red light centered on the wavelength        of 620 nm and assembled such that it illuminates the lower face        of the microplate homogenously,    -   a telecentric objective produced to make it possible to image        the entire surface of the microplate,    -   a filter wheel inserted between the output lens of the        telecentric objective and the camera,    -   a camera having the ability to produce images with exposure        times comprised between 0.001 second and 250 seconds,    -   a support chassis that supports and positions all of the        elements, including the microplate.        The optical bench is studied and assembled such that it takes        the images from the lower face of the microplate. The        development of the objective is done such that the inner face of        the microplate wells is clear.        The filter wheel is able to have two different filters:    -   a filter centered on the wavelength of 680 nm making it possible        to allow only the signal corresponding to the light emitted by        the fluorophore to pass,    -   a filter making it possible to allow all of the wavelengths        comprised between 400 nm and 700 nm to pass.        Description of the different steps carried out:        The test protocol comprises the following steps:

Step 1:

1. In each well of a microplate (comprising the spots) are successivelydistributed:+20 μl of diluent step 1+20 μl of diluent of conjugates 1 comprising the detection ligands ofthe analytes to be assayed from the first step.+40 μl of sample2. The mixture is incubated for 40 minutes at 37° C. with agitation.3. Three successive washes with at least 400 μl of wash solution aredone.

Step 2:

4. Distributed in each reaction well is 50 μl of diluent of conjugates 2containing the detection ligands of the analytes to be assayed from thesecond step.5. The mixture is incubated for 15 minutes at 37° C. with agitation.6. The wash steps (idem point 3) are carried out.

Step 3:

7. 50 μL of the S-POD reporter is distributed in each reaction well.8. The mixture is incubated for 15 minutes at 37° C. with agitation.

Step 4:

9. 25 μL of developing solution “B” is distributed in each reactionwell.10. 25 μL of developing solution “A” is distributed in each reactionwell.10. The mixture is incubated for 1 minute at 37° C. with agitation.11. The acquisition of the fluorescence signal is done for 10 seconds.12. The acquisition of the luminescence signal is done for 180 seconds.

Results

A) Persistence of the resistant control marker after an analysis method.

The fluorescence signal of the resistant control marker of each of the 9spots present in the 12 wells is clearly identifiable and fullymeasurable at the end of the analysis method in FIG. 2.

B) Importance of redefining the reading grid at the end of the analysismethod: comparison of the regions of interest obtained by fluorescenceby defining the reading grid relative to the theoretical positionsversus by defining the reading grid from the signal emitted byfluorescence by the resistant control marker.

In FIG. 3, the solid white circles show the theoretical position of thespots, the dotted white circles showing the detected actual position.The spots are clearly shifted relative to their expected theoreticalposition.

This image demonstrates the relevance of the method, which always makesit possible to target the position of the detected actual spot throughthe resistant control marker, which does not cause errors in the readingof the signal emitted by the detection marker of the detection ligand ofthe analyte.

C) Importance of redefining the reading grid at the end of the analysismethod: comparison of the regions of interest obtained bychemiluminescence by defining the reading grid relative to thetheoretical positions versus by defining the reading grid from thesignal emitted by fluorescence by the resistant control marker.

The location of the actual spots (dotted lines) was obtained based onsignals produced by the resistant control marker and applied on theacquisition image of the signals of the detection marker of a ligand ofthe analyte by chemiluminescence shown here in FIG. 4. The theoreticalpositioning of the spots is indicated in solid lines and clearly shows ashift relative to the actual position.

The comparison between the median values in chemiluminescence of thepixels situated under the expected theoretical positions (solid lines)and the actual detected positions (dotted lines) by fluorescence isshown in table 7.

TABLE 7 Signal measured from the analysis of the theoretical presenceregion of a spot versus signal measured from the analysis of the actualposition region of that same spot Spot 1 Spot 3 Spot 5 spot 9 (top left)(top right) (middle) (bottom right) Theoretical 319 301 58 3387 positionActual 689 693 138 5233 position

The analysis of the region containing a spot producing the signalprovides significantly higher results than the theoretical presenceregion of that same spot. The quantification of the signal and theaccuracy of the results obtained are therefore improved by basingoneself on the detected actual positioning grid owing to the resistantcontrol marker.

D) Example of deteriorated spot after an analysis method and that couldhave yielded a false result without verification of the integrity of thespots.

The location of the actual spots (dotted lines) was obtained based onsignals produced by the resistant control marker and applied on theacquisition image of the signals of the detection marker of a ligand ofthe analyte by chemiluminescence shown here (cf. FIG. 5). Thetheoretical positioning of the spots is indicated in solid lines.

In the image (cf. FIG. 5), the spot located at the center of the well isdeformed. The broken white line surrounding it shows that the softwaredetected the actual shape of the spot, which makes it possible toanalyze its integrity before validating the results yielded. In thiscase, the circularity perimeter of the actual shape of the spot makes itpossible to eliminate this spot and not yield a false analysis value.

We claim:
 1. A method for selecting a resistant control marker,comprising the following steps: a) depositing a marker to be tested onthe surface of a solid support, to form at least one spot, b)optionally, saturating the surface of the solid support, c) optionally,drying the surface of the solid support, d) carrying out at least one ofthe following steps: (i) performing one or more washing steps, (ii)placing the spot(s) in the presence of at least one detection ligand,(iii) placing the spot(s) in the presence of at least one reporter, (iv)placing the spot(s) in the presence of at least one substrate, e)selecting a marker that produces a signal at the end of step d).
 2. Amethod of using at least one resistant control marker in at least onespot intended to detect an analyte to secure a method for detecting atleast one analyte in a sample, characterized in that it comprises:checking the quality of said spot, after said spot has been placed inthe presence of the sample and at least one detection ligand of ananalyte to be detected, and/or the reading of the signal produced by atleast one detection marker of a detection ligand of an analyte to bedetected on a reading grid defined from the location of the signalproduced by said resistant control marker(s) and detected at the end ofthe analysis method.